U.S. patent application number 13/826804 was filed with the patent office on 2014-04-17 for adjuvant and antigen particle formulation.
The applicant listed for this patent is Flow Pharma, Inc.. Invention is credited to DAVID EARL HECKERMAN, CHARLES VINCENT TAYLOR HERST, REID M. RUBSAMEN.
Application Number | 20140105970 13/826804 |
Document ID | / |
Family ID | 50475519 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140105970 |
Kind Code |
A1 |
RUBSAMEN; REID M. ; et
al. |
April 17, 2014 |
ADJUVANT AND ANTIGEN PARTICLE FORMULATION
Abstract
A composition as disclosed is comprised of a plurality of
particles and a pharmaceutically acceptable carrier. The particles
are comprised of (1) an adjuvant; (2) a biocompatible polymer which
maybe a co-polymer such as PLGA, and (3) a peptide of a sequence of
interest, e.g. a sequence which corresponds to a sequence presented
on a surface of a cell infected with a virus. The carrier includes
an adjuvant such as a monophosphoryl lipid A (MPL) different from
the adjuvant in the particles. The particles may be sized such that
they are sufficiently large so as to prevent more than the contents
of a single particle from being presented to a single immune system
cell.
Inventors: |
RUBSAMEN; REID M.; (Alamo,
CA) ; HERST; CHARLES VINCENT TAYLOR; (Oakland,
CA) ; HECKERMAN; DAVID EARL; (Santa Monica,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Flow Pharma, Inc. |
East Palo Alto |
CA |
US |
|
|
Family ID: |
50475519 |
Appl. No.: |
13/826804 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13691234 |
Nov 30, 2012 |
|
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13826804 |
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61565686 |
Dec 1, 2011 |
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Current U.S.
Class: |
424/451 ;
424/184.1; 424/489 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/412 20180101; A61K 2039/55572 20130101; A61K 39/12
20130101; A61K 39/39 20130101; C12N 2760/20234 20130101; A61K
2039/55561 20130101; A61K 9/1647 20130101; A61K 9/16 20130101; A61K
2039/55555 20130101 |
Class at
Publication: |
424/451 ;
424/184.1; 424/489 |
International
Class: |
A61K 9/16 20060101
A61K009/16 |
Claims
1. A composition, comprising: a pharmaceutically acceptable carrier
comprising a first adjuvant; a group of particles comprising (a) a
biocompatible polymer; (b) an antigen; and (c) a second adjuvant
different from the first adjuvant; and wherein the particles are
substantially spherical, and have a diameter in a range of from 10
microns.+-.20% to 20 microns.+-.20%.
2. The composition as claimed in claim 1, wherein the carrier
comprises water and the first adjuvant is dissolved in the carrier,
and the second adjuvant is dispersed in the particles.
3. The composition as claimed in claim 1, wherein the first
adjuvant is a toll-like receptor agonist.
4. The composition as claimed in claim 3, wherein the toll-like
receptor agonist is a lipid polysaccharide (LPS).
5. The composition as claimed in claim 1, wherein the first
adjuvant is monophosphoryl lipid A (MPL) and the second adjuvant is
a single stranded DNA molecule.
6. The composition of claim 1, wherein the first adjuvant is
monophosphryl lipid A (MPL) and the second adjuvant is a CpG
oligodeoxynucleotide.
7. The composition of claim 1, wherein the antigen is an amino acid
sequence consisting of 8 to 12 amino acids and the biocompatible
polymer is selected from the group consisting of
poly(lactic-co-glycolic acid) (PLGA), polycaprolactone,
polyglycolide, polylactic acid, poly-3-hydroxybutyrate and the
first adjuvant is monophosphoryl lipid A (MPL) in the carrier, and
the second adjuvant is a CpG oligodeoxynucleotide in the
particles.
8. The composition, comprising: a pharmaceutically acceptable
aqueous carrier comprising a monophosphoryl-lipid-A (MPL) adjuvant;
and a group of substantially spherical particles having a diameter
in a range of about 10 microns.+-.20% to about 20 microns.+-.20%,
which particles are comprised of multiple copies of a class I
epitope and multiple copies of a class II epitope, a CpG
oligodeoxynucleotide adjuvant and a biocompatible polymer selected
from the group consisting of poly(lactic-co-glycolic acid) (PLGA),
polycaprolactone, polyglycolide, polylactic acid,
poly-3-hydroxybutyrate.
9. The composition of claim 1, wherein all the in particles of the
composition are 12 microns to 18 microns in diameter.+-.20% and the
first adjuvant is contained within the particles.
10. The composition of claim 1, wherein the antigen comprises (a)
multiple copies of a class I epitope and (b) multiple copies of a
class II epitope.
11. The composition of claim 1, wherein the antigen consisting only
of multiple copies of a class I epitope.
12. The composition of claim 10, wherein each class I epitope
consist of 8 to 20 amino acids.
13. The composition of claim 12, wherein each class I epitope
consists of 8 to 12 amino acids; and wherein the biocompatible
inert polymer is selected from the group consisting of
poly(lactic-co-glycolic acid) (PLGA), polycaprolactone,
polyglycolide, polylactic acid, poly-3-hydroxybutyrate.
14. The composition of claim 12, further comprising: (a) spherical
inert biocompatible particles; and (b) spherical inert
biocompatible particles encapsulating ink.
15. A composition, comprising: a pharmaceutically acceptable
carrier comprising a monophosphoryl lipid A (MPL) adjuvant; a group
of particles comprising (a) a biocompatible polymer; (b) an
antigen; and (c) an adjuvant, different from the (MPL); and wherein
the particles are substantially spherical, and have a diameter in a
range of from 10 microns.+-.20% to 25 microns.+-.20%.
16. The composition of claim 15, wherein all the particles of the
composition are 12 microns to 22 microns in diameter.+-.20% and the
adjuvant different from the (MPL) is a CpG
oligodeoxynucleotide.
17. A method of treatment, comprising: administering to a human
patient a composition comprising: a pharmaceutically acceptable
carrier comprising a monophosphoryl lipid A (MPL) adjuvant; a group
of particles comprising (a) a biocompatible polymer; (b) an
antigen; and (c) a CpG oligodeoxynucleotide adjuvant; and wherein
the particles are substantially spherical, and have a diameter in a
range of from 10 microns.+-.20% to 25 microns.+-.20%.
18. The method of claim 17, further comprising: administering to
the patient an ink visible on skin of the patient.
19. A composition, comprising: a plurality of particles comprised
of a biocompatible polymer, an antigen, and a CpG
oligodeoxynucleotide adjuvant; and a pharmaceutically acceptable
aqueous carrier comprised of a monophosphoryl lipid A (MPL)
adjuvant.
20. The composition of claim 19, wherein the particles are
substantially spherical, and have a diameter in a range of from 10
microns.+-.20% to 25 microns.+-.20%.
Description
CROSS-REFERENCE
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 13/691,234, filed Nov. 30, 2012 which
application claims the benefit of U.S. Provisional Application No.
61/565,686, filed Dec. 1, 2011.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of
pharmaceutical formulations and more particularly to vaccine
formulations comprised of two adjuvants.
BACKGROUND OF THE INVENTION
[0003] The term vaccine derives from Edward Jenner's 1796 use of
the term cow pox (Latin variol.ae butted. vaccin.ae butted.,
adapted from the Latin vacc n-us, from vacca cow), which, when
administered to humans, provided them protection against
smallpox.
[0004] The 20th century saw the introduction of several successful
vaccines, including those against diphtheria, measles, mumps, and
rubella. Major achievements included the development of the polio
vaccine in the 1950s and the eradication of smallpox during the
1960s and 1970s. Maurice Hilleman was the most prolific of the
developers of the vaccines in the twentieth century. As vaccines
became more common, many people began taking them for granted.
However, vaccines remain elusive for many important diseases,
including malaria and HIV. Vaccines may be dead or inactivated
organisms or purified products derived from them.
[0005] There are several types of vaccines currently in use. These
represent different strategies used to try to reduce risk of
illness, while retaining the ability to induce a beneficial immune
response. Considerable efforts have been made to develop an HIV
vaccine.
[0006] A cell infected with HIV virus has many distinct epitopes on
its surface. Each epitope can be targeted by the cellular immune
response mediated by T-lymphocytes. These T-lymphocytes become
sensitized to specific epitopes by exposure to antigens brought to
the T-cells by antigen presenting cells (e.g macrophages).
[0007] HIV vaccines have been developed to direct cellular immunity
mechanisms toward a blood borne HIV virus by sensitizing T-cells,
via antigen presenting cells (APCs) exposed to the vaccine, to
suites of epitopes on the surface of cells infected with the
virus.
[0008] Vectors used to introduce vaccines into the cellular
immunity pathways have included adenovirus vectors. A problem with
traditional vaccine approaches to treating patients already
infected with HIV has been the fact that adenovirus vectors tend to
activate CD4.sup.+ T-cells which in turn can potentially make
pre-existing HIV infection more virulent. Another problem with HIV
vaccine designs, in general, has been that the end result is to
target large suites of epitopes on the surface of the cells
infected with the virus, possibly targeting epitopes which could
actually worsen various pathological aspects of the HIV
infection.
[0009] Until recently, little was known about the specific effect
of targeting specific epitopes on the surface of cells infected
with the HIV virus. Each time a vaccine vector is given to a person
with HIV disease, a number of epitopes are targeted, and a number
of immune response parameters are measured. Associating a specific
response with a specific epitope has been essentially impossible
from an analysis of a single vaccine administration. Data from the
administration of multiple vaccines to multiple sets of HIV
infected subjects with corresponding cellular immune responses
could, in theory, allow the effects of the individual epitopes to
be de-convolved, essentially through a very computational intensive
cross-correlation exercise.
[0010] Recent work in the field has brought modern super-computer
technology to bear on this problem, resulting in a list of putative
individual pathogen-relevant effects of individual epitopes on the
surface of cells infected with the HIV virus.
[0011] Results from immunization with antigen-containing PLGA
microspheres made from a double-emulsion process utilizing organic
solvents have been mixed, however, perhaps owing to the fact that
the solvent systems and shear forces used in such microsphere
fabrication processes can cause protein conformational changes that
may interfere with the antigen-presenting event.
[0012] Peptides injected into the lymphatic system can be taken up
by APCs, thereby producing an immune response. If a single APC
takes up more than one antigen and simultaneously presents multiple
antigens to T-cells, this may result in a cellular immune response
wherein the response is targeted to only one of the antigens that
was presented.
[0013] Methods for relieving the effects of immunodominance are
described in published US patent application 20080260780, entitled
"Materials And Methods Relating To Improved Vaccination
Strategies"; US patent application 20090269362, entitled "Method
for Controlling Immunodominance"; and US patent application
20100119535, entitled "Compositions and Methods for Immunodominant
Antigens."
SUMMARY OF THE INVENTION
[0014] A composition is disclosed which is comprised of a carrier;
adjuvants, and a group of particles, which may be substantially
spherical particles, comprised of a polymer and an antigen, e.g. a
peptide, lipid, glycolipid, phospholipid, polysaccharide, etc. One
adjuvant may be inside and/or on the surface of the particles and
one adjuvant in the carrier.
[0015] All of the particles in all of the groups have a narrow
particle size range of about 10 .mu.m.+-.20% to about 20
.mu.m.+-.20% in diameter. The size range can be such that only a
single particle can be consumed by an antigen presenting cell, by
which is usually intended a professional antigen presenting cell.
For example, the particle size may be in a range of 12 microns to
18 microns in diameter. The particles sizes may be 15 microns in
diameter.+-.20%. The size of the particles in a given formulation
may be substantially the same size, i.e. .+-.20% or .+-.10%,
.+-.2%, or .+-.1% in diameter.
[0016] One embodiment of the formulation uses a particular
combination of adjuvants where a monophosphoryl lipid A (MPL)
adjuvant is within and/or on the surface of particles and a CpG
oligodeoxynucleotide is part of the carrier. The combination of
adjuvants with the closely controlled particle size produces a
robust immune response.
[0017] Each particle comprises one or more antigenic compounds,
including without limitation peptides, where the antigenic
compounds in a particle may be of identical species or sequence.
When peptides are used they are generally small and should be at
least 8 and not more than 20 amino acids, not more than 15, or not
more than 11 or 12 amino acids in length. Thus, the peptide size
range may be 8-12 amino acids particularly epitopes for
presentation by class I MHC proteins. However, where epitopes for
presentation by class II MHC proteins are present, the peptides may
have a broader size range, e.g. 8 or more amino acids in length, or
8-30 amino acids.
[0018] In one embodiment of the invention, peptides present in or
on any one particle may be identical to all other peptides on that
particle, i.e. the peptides have the same amino acid sequence and
there may be no other peptides in that particle with a different
amino acid sequence; other compounds such as lipids,
polysaccharides, etc. consist of a single species. In another
embodiment, a particle comprises (i) one or more species of peptide
that provides an epitope for class I presentation, and (ii) one
species of peptide that provides an epitope for class II
presentation. In certain of such embodiments, a given particle is
comprised of an inert polymer such as PEG, and comprises two or
more different species of peptides where one species provides a
class I epitope that generates a specific immune response and the
other peptide species provides a class II epitope that enhances or
amplifies the immune response generated by the class I epitope.
[0019] The particles may be provided as a group, i.e. a composition
or formulation of a plurality of particles with an adjuvant and an
antigen. Alternatively a composition or formulation is provided
comprising a cocktail of particles with a plurality of different
adjuvant and antigen species.
[0020] In one embodiment the composition comprises an additional
adjuvant in a form chosen from (a) inert biocompatible particles
with no other components inside; (b) inert biocompatible particles
with only ink inside; and (c) inert biocompatible particle with a
peptide presented by a class II peptide inside.
[0021] In another embodiment of the invention a conventional
vaccine formulation is combined with any or all of the adjuvant
(a), (b) and/or (c) above and administered with a conventional
adjuvant.
[0022] Formulations of the invention can be administered as
vaccines to prevent an infection or as a treatment for a
disease.
[0023] Formulations of the invention may be administered by any
type of injection or nasally.
[0024] Formulations of the invention can act as B-cell vaccines as
well as T-cell vaccines.
[0025] Compositions or groups of interest for manufacturing
purposes, in kits, and the like may comprise a carrier with an
adjuvant, and particles containing a different adjuvant along with
one or more antigen species. Compositions and formulations for
therapeutic purposes, e.g. for use in a method of treatment,
generally include a plurality of species. Formulations for
generating an immune response may include 2, 3, 4, 5, 10, 20, 30,
40, 50 or more antigen species and an equivalent number of groups
of particles.
[0026] The composition containing the plurality of groups of
particles may be a vaccine formulation which may have the particles
in a dry form to which a liquid carrier can be added to form a
suspension or emulsion (e.g. injectable or viscous nasal spray), or
the liquid carrier may be present and the liquid carrier may be a
pharmaceutically acceptable injectable carrier.
[0027] A composition of the invention can include any combination
of the compositions outlined in the claims below. A particular
composition of the invention which includes the components of the
first seven claims below would be a composition which reads as
follows:
[0028] A composition, comprising: [0029] a group of particles
comprising (a) a biocompatible polymer; (b) an antigen; and (c) a
monophosphoryl lipid A (MPL); and [0030] a pharmaceutically
acceptable carrier comprising a CpG oligodeoxynucleotide; [0031]
wherein the particles are substantially spherical, and have a
diameter in a range of from 10 microns.+-.20% to 25
microns.+-.20%.
[0032] The compositions of the invention can include a composition
as described above wherein the adjuvant includes toll-like receptor
agonists which include CpG and MPL which is monophosphoryl lipid A.
The term CPG is further described below.
[0033] CpG oligodeoxynucleotides (or CpG ODN) are short
single-stranded synthetic DNA molecules that contain a cytosine "C"
followed by a guanine "G". The "p" refers to the phosphodiester
backbone of DNA, however some ODN have a modified phosphorothioate
(PS) backbone. When these CpG motifs are unmethlyated, they act as
immunostimulants. CpG motifs are considered pathogen-associated
molecular patterns (PAMPs) due to their abundance in microbial
genomes but their rarity in vertebrate genomes. The CpG PAMP is
recognized by the pattern recognition receptor (PRR) Toll-Like
Receptor 9 (TLR9), which is constitutively expressed only in B
cells and plasmacytoid dendritic cells (pDCs) in humans and other
higher primates.
[0034] The toll-like receptors can stimulate antigen presentation
by dendrocytes. Toll receptors such as MPL are less active versions
of LPS which is a lipid polysaccharide. The LPS causes signs and
symptoms of sepsis. The LPS promotes phagocytosis. An aspect of the
invention includes incorporating CpG inside the sphere and placing
the MPL in solution which the spheres are suspended in as described
in the examples. The CpG is included inside the cell in order to
promote antigen presentation once the sphere has been
phagosytosed.
[0035] An aspect of the invention is the formulation for delivering
the adjuvant and peptide containing microspheres as described here
for vaccination where a toll-like receptor agonist to promote
antigen presentation is incorporated into the sphere and a
toll-like receptor agonist to promote phagocytosis is added to the
solution in which the spheres are suspended.
[0036] In another aspect of the invention such a formulation is
produced wherein the toll-like receptor agonist to promote antigen
presentation is CpG in the sphere and a toll-like receptor agonist
to promote phagocytosis is MPL.
[0037] Another aspect of the invention is a nasal formulation
containing compositions as encompassed by the claims. Such nasal
formulations for the delivery of vaccines can be produced using
excipient materials known to those skilled in the art such as
disclosed within U.S. Pat. No. 6,391,318 (incorporated herein by
reference) as well as the patents and publications cited in the
'318 patent all of which are incorporated herein by reference.
[0038] The invention also includes a method of treating a subject
such as vaccinating a subject whereby a formulation of the
invention is injected into an individual in order to elicit an
immune response.
[0039] The method of the invention comprises administering to a
human patient a composition comprising a group of particles
comprising an adjuvant, a biocompatible polymer and a first
chemical species, and a second adjuvant which may act as a
pharmaceutically acceptable carrier for the particles. The
particles are substantially spherical in shape and have a diameter
of from 10 microns.+-.20% to 25 microns.+-.20% or 12 microns to 22
microns in diameter.+-.20% or about 17 microns in diameter.+-.20%.
The method is comprises administering the composition which may be
the initial exposure of the patient to the chemical species or
antigens. More specifically, the patient has not been previously
exposed to such antigens. Thus, the method of the invention is not
intended to be a vaccination booster within a more general method
where the patient has previously been primed or exposed to a
plurality of epitopes. Thus, the compositions of the invention are
generally not intended for use as a "booster" to supplement the
immune response of an earlier exposure to an antigen or plurality
of antigens.
[0040] An aspect of the invention is that antigen presenting cells
take up a single particle of the invention, which particle may
comprise a single antigenic species; or a single species that
provides an epitope for class I presentation. When only a single
antigenic species is present, problems with immunodomination may be
reduced or eliminated. Accordingly, the immune system will produce
a plurality of immune responses (e.g., multiple cytotoxic T-cell
clones; antibodies; etc.) in response to the different antigenic
species presented in different particles. This end result can be
achieved by using a formulation as described here, which is
comprised of groups of particles wherein each group of particles
consists of only one type of antigenic particle. The result can
also be obtained by a methodology whereby a formulation consisting
of a first antigenic compound which is injected at a first point in
time followed by an injection of a formulation consisting of a
second antigenic compound at a second point in time after the first
point in time. Additional steps in the process may be carried out
with a third, fourth, fifth, etc. antigenic compound administered
at a third, fourth, fifth, sixth, etc. point in time. The end
result is to obtain multiple different immune responses with
multiple different cytotoxic T-cells or multiple different
antibodies which bind to multiple different antigens and thereby
reduce or eliminate the immunodominance issue with respect to a
human immune system. This methodology requires multiple
administrations which may not be required when the formulation is
comprised of several different groups of particles wherein each
particle consists of only a single antigenic compound. Either the
formulation of multiple groups or the step by step methodology
using one group with each administration can be used to achieve a
basic result whereby only a single antigenic compound is presented
to a given cell at a given point in time to thereby reduce or
eliminate the immunodominance problem resulting from simultaneous
presentation of more than one antigenic compound at the same
time.
[0041] The invention also includes a method of making the particles
and the population of groups of particles in order to form the
formulation.
[0042] Formulations used to generate an immune response such as
vaccine formulations of the invention can comprise polypeptides
that correspond to an epitope, usually a T cell epitope, that, when
brought into contact with a mammalian (e.g. human) immune system
will elicit an immune response targeted to the corresponding
epitope on a cell, virus, etc. Epitopes of interest include,
without limitation, those present on pathogens, e.g. virus,
bacteria, fungi, protozoan, etc.; and may further comprise epitopes
associated with, for example, cancer cells. The polypeptides are
encapsulated within and/or bound to the surface of specifically
sized particles, where the particles may be formed of any suitable
biocompatible material, e.g. biocompatible polymers such as
poly(lactic-co-glycolic acid) (PLGA), polycaprolactone,
polyglycolide, polylactic acid, poly-3-hydroxybutyrate, etc.
[0043] The invention provides optimally sized particles comprised
of a biocompatible polymer, wherein the particles are only loaded
with an adjuvant and a population of peptides, where the peptides
may correspond to a single epitope, and further wherein the
particles are sized so that only a single particle is taken into a
single APC at a given point in time.
[0044] In another aspect of the invention the peptides consisting
only of 8 to 20 amino acids; or 8 to 15 amino acids with no other
antigenic compound present.
[0045] In yet another aspect of the invention the composition
further comprises a third, and possibly further additional groups
of particles (4, 5, 6 . . . 10 . . . 20 . . . 30, etc. groups)
wherein each group is comprised of an adjuvant, a biocompatible
polymer and an antigen chemical species.
[0046] In another aspect of the invention the biocompatible polymer
is a polymer selected of the group consisting of
poly(lactic-co-glycolic acid) (PLGA), polycaprolactone,
polyglycolide, polylactic acid, poly-3-hydroxybutyrate.
[0047] Another aspect of the invention is a use of a composition as
described here in the manufacture of a formulation for treatment of
a patient in order to generate an immune response within the
patient.
[0048] Another aspect of the invention is a method of treatment
which may be a method of generating an immune response in a subject
by administering to a subject a formulation as described and
disclosed herein and thereafter allowing the formulation to
interact with the subject's immune system and thereby generate an
immune response.
[0049] In another aspect of the invention the formulation is
administered by injection.
[0050] In yet another aspect of the invention the formulation is
administered by contacting the formulation with nasal membranes of
the subject such as by the use of a nasal spray, nasal drops, or
other formulation designed for administration to and contact with a
subject's nasal membrane wherein the formulation may have a
viscosity several times (2, 3, 4, etc) more viscous than water to
reduce dripping of the formulation from the nasal cavity.
[0051] Another aspect of the invention is to provide a process
whereby the particles as described above are formed by extrusion
from a nozzle in a manner which creates particles and does not
damage the adjuvant or antigenic compound, e.g. peptides, etc
thereby allowing each component to substantially maintain its
antigenic property.
[0052] Another aspect of the invention is to provide such a process
for producing particles which can be carried out without the use of
solvents including organic solvents or any other compounds beyond
the biocompatible polymer and chemical species.
[0053] Also disclosed is a method of treatment, comprising: [0054]
administering to a subject a formulation comprising: [0055] a group
of particles comprising (a) a biocompatible polymer; (b) an
antigen; and (c) a monophosphoryl lipid A (MPL); and [0056] a
pharmaceutically acceptable carrier comprising a CpG
oligodeoxynucleotide;
[0057] wherein the particles are substantially spherical, and have
a diameter in a range of from 10 microns.+-.20% to 25
microns.+-.20%; [0058] wherein the antigen consists of 8 to 15
amino acids; and [0059] allowing the formulation to interact with
the subject's immune system and thereby generate an immune
response.
[0060] Another aspect on the invention is a method as recited
above, wherein the formulation is administered by injection into
the subject's lymph system.
[0061] Another aspect on the invention is a method as recited
above, wherein the amino acid sequences comprise epitopes of a
pathogen or epitopes on the surface of a cell infected with a
pathogen.
[0062] Another aspect on the invention is a method as recited
above, wherein the pathogen is selected from the group consisting
of a virus, a bacteria and a parasite.
[0063] Another aspect on the invention is a method as recited
above, wherein the administering is by injecting the subject
formulation with the formulation.
[0064] Another aspect on the invention is a method as recited
above, wherein the administering is by contacting nasal membranes
of the subject with the formulation which may be a viscous or
sticky formulation designed for nasal administration, which
includes a high viscosity carrier (5, 10 or more times the
viscosity of water at 37.degree. C.) or spheres coated with a
bioadhesive.
[0065] An aspect of the invention is a vaccine formulation of the
invention further comprising ink which would mark skin of a human
patient as having been vaccinated.
[0066] Yet another aspect of the invention is an ink vaccine
formulation wherein the ink is visible only under a black
light.
[0067] Still another aspect of the invention is a vaccine
formulation wherein the ink is encapsulated in microspheres wherein
the microspheres are comprised of a biocompatible polymer and are 5
.mu.m to 20 .mu.m in diameter.
[0068] Still yet another aspect of the invention is a vaccine
system whereby the ink in the vaccines is used to individually
categorize the human patients vaccinated by imprinting a unique
mark (e.g. number, letter, symbol or combination thereof) on each
human patient vaccinated.
[0069] An aspect of the invention includes administering any
formulation of the invention using a needle device conventionally
used to draw a tattoo on a patient.
[0070] These and other objects, advantages, and features of the
invention will become apparent to those persons skilled in the art
upon reading the details of the formulations and methods of
treatment as more fully described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIG. 1 includes four photos (1A, 1B, 1C and 1D). Each of the
photos show human dendrocytes and 10 micron diameter particles
comprised of PLGA. In FIG. 1A two human dendrocytes are shown in
proximity to the microspheres. In FIG. 1B a first human dendrocyte
is shown phagocytosing three 10 micron PLGA microspheres with a
second dendrocyte beginning phagocytosis. In FIG. 1C the first
dendrocyte is shown completing phagocytosis of three 10 micron PLGA
microspheres and a second dendrocyte in active phagocytosis. In
FIG. 1D the second dendrocyte has completed phagocytosis of three
10 micron PLGA microspheres.
[0072] FIG. 2 includes four photos (2A, 2B, 2C and 2D) each of
which show human dendrocytes and PLGA microspheres which are 15
microns in diameter. In FIG. 2A two human dendrocytes are in
proximity with two 15 micron microspheres. In FIG. 2B a first
dendrocyte is phagocytosing a 15 micron microsphere. In FIG. 2C a
first dendrocyte has undergone complete phagocytosis of only a
single 15 micron microsphere and a second dendrocyte is in active
phagocytosis. In FIG. 2D a second dendrocyte has completed
phagocytosis of a single 15 micron microsphere.
[0073] These still photos from FIGS. 1 and 2 were taken from videos
of human dendrocytes placed in proximity to microspheres wherein
the microspheres were 10 microns in diameter for FIG. 1 and 15
microns in diameter for FIG. 2. The photos are part of many taken
and were chosen from many other photos as photos which might best
represent aspects of the invention.
[0074] FIG. 3 shows three images which schematically represent
particles of the invention. In FIG. 3A the particles include a 1:1
ratio of a first antigen OVA to a second antigen VSV. FIG. 3B shows
a schematic image of a particle of the invention wherein the ratio
of the OVA antigen to the VSV antigen is 1:10. In FIG. 3C the ratio
of the OVA antigen to the VSV antigen is 1:100.
[0075] FIG. 4 includes three sets of images. In FIG. 4A the images
schematically represent a first group of particles which contain a
first antigen OVA with a second image representing a second group
of particles with a second antigen VSV wherein the first group of
particles and second group of particles include a single antigen
per particle. FIG. 4B includes two images wherein the first image
represents a particle of the invention including a single antigen
of OVA and the second image represents a particle which includes 10
antigens of VSV. FIG. 4C includes two images wherein the first
image represents a group of particles wherein the particles include
a single antigen of OVA and a second image which includes a group
of particles wherein each particle includes 100 copies of the
antigen VSV.
[0076] FIG. 5 shows a graph representing ratios of the data
obtained using the particles in FIGS. 3 and 4. The graph of FIG. 5
is based on three data points wherein data point 1 is the ratio of
the immune response obtained with particles of FIG. 3A relative to
the immune response obtained with particles of FIG. 4A. Data point
2 is the ratio of the immune response obtained with particles of
FIG. 3B relative to the immune response obtained with particles of
FIG. 4B. Data point 3 is the ratio of the immune response obtained
with particles of FIG. 3C relative to the immune response obtained
with the particles of FIG. 4C. The results of FIG. 5 show that by
separating antigens into two different groups of particles wherein
the particles within each group include a different antigen from
the other group a greater immune response is obtained.
[0077] FIG. 6 shows a plot and data related to Example 6 showing
particle size distribution indicating highly uniform particles
sizes.
[0078] FIG. 7A shows a graph demonstrating the effect of adding the
adjuvant CpG to OVA loaded spheres which are 10 microns in
diameter. Two groups of mice were immunized with 10 micron
OVA-loaded microspheres in a carrier solution containing MPLA with
and without CpG added to the sphere. The increase in the immune
response seen in FIG. 7A after the addition of CpG to the sphere is
statistically significant (p=0.0002). FIG. 7B is a chart showing
the mean, standard deviation, standard error of mean for the data
collected. FIG. 7C is a chart showing the number of data points
analyzed and the outliers.
[0079] FIG. 8A is a graph demonstrating the effect of placing a
monophosphoryl lipid A (MPL) adjuvant inside 10 micron spheres as
compared to placing the same adjuvant in a carrier solution used as
a carrier for the spheres. Two groups of mice were immunized in
order to demonstrate the effect of MPLA in the carrier solution
versus 10 micron OVA-loaded microspheres with CpG. The improvement
in the immune response is demonstrated with the MPLA in the carrier
solution and the improvement is statistically significant
(p=0.0002). FIG. 8B is a chart showing the mean, standard deviation
and standard error of mean for the data collected. FIG. 8C is a
chart showing the number of data points analyzed and the number of
outliers.
DETAILED DESCRIPTION OF THE INVENTION
[0080] Before the present composition, formulation and method of
manufacture and use and treatment are described, it is to be
understood that this invention is not limited to particular
embodiment described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0081] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0082] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0083] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DEFINITIONS
[0084] A vaccine is a biological preparation intended to improve a
recipient's immunity to a particular disease. A vaccine typically
contains an agent that resembles a disease-causing microorganism,
and is often made from weakened or killed forms of the microbe or
its toxins. The agent stimulates the body's immune system to
recognize the agent as foreign, destroy it, and "recognize" it, so
that the immune system can more easily recognize and destroy any of
these microorganisms that it later encounters. Vaccines can be
prophylactic (e.g. to prevent or ameliorate the effects of a future
infection by any natural or "wild" pathogen), or therapeutic (e.g.
vaccines against cancer are also being investigated).
[0085] The expression "enhanced immune response" or similar term
means that the immune response is elevated, improved or enhanced to
the benefit of the host relative to the prior immune response
status, for example, a native status before the administration of
an immunogenic composition of the invention or to obtained a
stronger immune response relative to the response in the absence of
enhancement, e.g. 10%, 20% . . . 100% enhanced.
[0086] The terms "cell-mediated immunity" and "cell-mediated immune
response" are meant to refer to the immunological defense provided
by lymphocytes, such as that defense provided by T cell lymphocytes
when they come into close proximity to a target cell. A
cell-mediated immune response normally includes lymphocyte
proliferation. When "lymphocyte proliferation" is measured, the
ability of lymphocytes to proliferate in response to a specific
antigen is measured. Lymphocyte proliferation is meant to refer to
T-helper cell or cytotoxic T-lymphocyte (CTL) cell
proliferation.
[0087] The term "immunogenic amount" refers to an amount of
antigenic compound sufficient to stimulate an enhanced immune
response, when administered with a subject immunogenic composition,
as compared with the immune response elicited by the antigen in the
absence of the microsphere formulation.
[0088] The terms "treatment", "treating", "treat" and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect such as an enhanced immune response. The
effect may be prophylactic in terms of completely or partially
preventing a disease or symptom thereof and/or may be therapeutic
in terms of a partial or complete stabilization or cure for a
disease and/or adverse effect attributable to the disease.
"Treatment" as used herein covers stimulating an immune response,
and any treatment of a disease in a subject, particularly a
mammalian subject, more particularly a human, and includes: (a)
preventing the disease or symptom from occurring in a subject which
may be predisposed to the disease or symptom but has not yet been
diagnosed as having it; (b) inhibiting the disease symptom, e.g.,
arresting its development; or relieving the disease symptom, i.e.,
causing regression of the disease or symptom (c) reduction of a
level of a product produced by the infectious agent of a disease
(e.g., a toxin, an antigen, and the like); and (d) reducing an
undesired physiological response to the infectious agent of a
disease (e.g., fever, tissue edema, and the like).
[0089] The term "antigen presenting cell" or APC may generally
refer to a mammalian cell having a surface HLA Class I or HLA Class
II molecule in which an antigen is presented. Unless otherwise
indicated, for the purposes of the present invention an antigen
presenting cell is a "professional" antigen presenting cell that
can activate or prime T cells, including naive T cells.
Professional APC usually express both HLA Class I and HLA Class II
molecules, and are very efficient at internalizing antigens, either
by phagocytosis or by receptor-mediated endocytosis, and then
displaying an antigen or a fragment thereof bound to the
appropriate HLA molecule on their cell surface. Synthesis of
additional co-stimulatory molecules is a defining feature of
professional APCs. Of these APCs, dendritic cells (DCs) have the
broadest range of antigen presentation, and are the most important
T cell activators. Macrophages, B cells and certain activated
epithelial cells are also professional APCs.
[0090] An adjuvant of the invention is an agent that may stimulate
the immune system and increase the response to the antigen
component without having any specific antigenic effect in itself.
The adjuvant may be any substance that acts to accelerate, prolong,
or enhance antigen-specific immune responses when used in
combination with a specific antigen component of the invention. The
invention shows that the physical location of the adjuvant relative
to the shape of the sphere is important in allowing the adjuvant to
generate an immune response.
[0091] FIG. 7A shows that an improved immune response can be
obtained by placing a CpG oligodeoxynucleotide (CpG) inside 10
micron spheres. Data relating to the graphs of 7A is shown in FIGS.
7B and 7C.
[0092] The graph of FIG. 8A shows that a substantially improved
immune response can be obtained by placing a monophosphoryl lipid A
(MPL) in the solution containing the spheres as compared to placing
the same adjuvant in the spheres themselves. The data used in a
preparation of FIG. 8A is described further within the charts of
FIGS. 8B and 8C.
[0093] When a monophospholipid A (MPLA) adjuvant was administered
it was administered to mice in the amount of 10 micrograms per
mouse. When the adjuvant was administered in a carrier solution, 10
micrograms was present in a 100 microliter injection for each
mouse. If the adjuvant was present in microparticles, the 10
micrograms of adjuvant was present in 2 milligrams of
microparticles injected into each mouse. This adjuvant could be
administered to each mouse in an amount in a range of about 5
micrograms to 20 micrograms. When the mice were treated with the
CpG adjuvant the mice were administered 5 micrograms of the
adjuvant which was present within 2 milligrams of particles
injected into each mouse.
[0094] Those skilled in the art will understand that the amounts
described above can vary substantially over a range and still
obtain a therapeutic effect and remain below a toxic level.
Further, those skilled in the art will understand that the amounts
given above are amounts with respect to mice and those amounts can
be readily extrapolated with respect to humans. Further, the dosing
can be adjusted based on the age, sex, weight, condition and other
factors of the patient being treated.
[0095] There are many known adjuvants in widespread use, including
oils, aluminium salts, and virosomes. Adjuvants may provide
increased immunity to a particular disease. Adjuvants accomplish
this task by mimicking specific sets of evolutionarily conserved
molecules, so called PAMPs, which include liposomes,
lipopolysaccharide (LPS), molecular cages for the antigen,
components of bacterial cell walls, and endocytosed nucleic acids
such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA),
and unmethylated CpG dinucleotide-containing DNA. Because immune
systems have evolved to recognize these specific antigenic
moieties, the presence of an adjuvant in conjunction with the
antigen can greatly increase the innate immune response to the
antigen by augmenting the activities of dendritic cells (DCs),
lymphocytes, and macrophages by mimicking a natural infection.
Furthermore, because adjuvants are attenuated beyond any function
of virulence, they pose little or no independent threat to a host
organism.
[0096] There are many adjuvants, some of which are inorganic (such
as alum), that also carry the potential to augment immunogenicity.
Two common salts include aluminium phosphate and aluminium
hydroxide. These are the most common adjuvants in human
vaccines.
[0097] While Aluminium salts are popularly used in human vaccines,
the organic compound squalene is also used (e.g. AS03). However,
organic adjuvants are more commonly used in animal vaccines.
Oil-based adjuvants are commonly used in some veterinary vaccines.
MF59 is an `oil [squalene] in water` adjuvant used in some human
vaccines.
[0098] Other market-approved adjuvant and carrier system include
virosomes. During the last two decades, a variety of technologies
have been investigated to improve the widely-used adjuvants based
on aluminium salts. These salts are unfavorable, since they develop
their effect by inducing local inflammation, which is also the
basis for the extended side-effect pattern of this adjuvant. In
contrast, the adjuvant capabilities of virosomes are independent of
any inflammatory reaction. Virosomes contain a membrane-bound
hemagglutinin and neuraminidase derived from the influenza virus,
and serve to amplify fusogenic activity and therefore facilitate
the uptake into antigen presenting cells (APC) and induce a natural
antigen-processing pathway. The delivery of the antigen by
virosomes to the immune system in a way that mimics a natural path
may be a reason why virosome-based vaccines stand out due to their
excellent safety profile.
[0099] One misconception concerning adjuvant function is that an
adjuvant-enhanced innate immune response should affect only the
transient reaction of the innate immune response and not the more
long-lived effects of the adaptive immune response. Although it may
appear fitting to separate the two systems, it is however important
to realize the interconnected nature of the two systems. When the
amount of communication that takes place between the innate immune
response and the adaptive immune response with the onset of
infection is considered it becomes difficult to separate the two
systems. Actual experimentation conducted with adjuvants shows that
improved results can be obtained (a) by using two different types
of adjuvants and (b) by physically placements of the adjuvants
relative to each other.
[0100] Differences in the results obtained using different
adjuvants in different physical places is dramatically seen in
FIGS. 7A and 8A. The CpG adjuvant has a dramatic improvement on the
immune response when included in the spheres themselves. However,
the MPLA adjuvant does not show a dramatic improvement when this
adjuvant is included in the spheres themselves while a dramatic
improvement is seen when the MPLA adjuvant is included in the
carrier solution in which the spheres are placed.
[0101] In order to understand the links between the innate immune
response and the adaptive immune response to help substantiate an
adjuvant function in enhancing adaptive immune responses to the
specific antigen of a vaccine, the following points should be
considered: [0102] Innate immune response cells such as Dendritic
Cells (DCs) engulf pathogens through a process called phagocytosis.
[0103] The phagocytosis is enhanced by placing an MPLA adjuvant in
a carrier for the spheres. [0104] After phagocytosis the DCs then
migrate to the lymph nodes where T cells (adaptive immune cells)
wait for signals to trigger their activation. [0105] In the lymph
nodes, DCs mince the engulfed pathogen and then express the
pathogen clippings as antigen on their cell surface by coupling
them to a special receptor known as a major histocompatibility
complex (MHC). [0106] T cells can then recognize these clippings
and undergo a cellular transformation resulting in their own
activation. [0107] T cells possess characteristics of both the
innate and adaptive immune responses. [0108] Macrophages can also
activate T cells in a similar approach (but do not do so
naturally).
[0109] This process carried out by both DCs and macrophages is
termed antigen presentation and represents a physical link between
the innate and adaptive immune responses.
[0110] Upon activation, mast cells release heparin and histamine to
effectively increase trafficking to and seal off the site of
infection to allow immune cells of both systems to clear the area
of pathogens. In addition, mast cells also release chemokines which
result in the positive chemotaxis of other immune cells of both the
innate and adaptive immune responses to the infected area.
[0111] Due to the variety of mechanisms and links between the
innate and adaptive immune response, an adjuvant-enhanced innate
immune response results in an enhanced adaptive immune response.
Specifically, a recent study has observed that adjuvants may exert
their immune-enhancing effects according to five immune-functional
activities. [0112] First, it was found that adjuvants all help in
the translocation of antigens to the lymph nodes where they can be
recognized by T cells. This will ultimately lead to greater T cell
activity resulting in a heightened clearance of pathogen throughout
the organism. [0113] Second, adjuvants provide physical protection
to antigens which grants the antigen a prolonged delivery. This
means that the organism will be exposed to the antigen for a longer
duration, making the immune system more robust as it makes use of
the additional time by up-regulating the production of B and T
cells needed for greater immunological memory in the adaptive
immune response. [0114] Third, adjuvants help to increase the
capacity to cause local reactions at the injection site (during
vaccination), inducing greater release of danger signals by
chemokine releasing cells such as helper T cells and mast cells.
[0115] Fourth, they induce the release of inflammatory cytokines
which helps to not only recruit B and T cells at sites of infection
but also to increase transcriptional events leading to a net
increase of immune cells as a whole. [0116] Finally, adjuvants are
believed to increase the innate immune response to antigen by
interacting with pattern recognition receptors (PRRs), specifically
Toll-like receptors (TLRs), on accessory cells.
[0117] The ability of the immune system to recognize molecules that
are broadly shared by pathogens is, in part, due to the presence of
special Immune receptors called TLRs that are expressed on
leukocytemembranes. TLRs were first discovered in drosophila, and
are membrane bound pattern recognition receptors (PRRs) responsible
for detecting most (although certainly not all) antigen-mediated
infections. In fact, some studies have shown that in the absence of
TLR, leukocytes become unresponsive (no inflammatory responses) to
some microbial components such as LPS. There are at least thirteen
different forms of TLR, each with its own characteristic ligand.
Prevailing TLR ligands described to date (all of which elicit
adjuvant effects) include many evolutionarily conserved molecules
such as LPS, lipoproteins, lipopeptides, flagellin, double-stranded
RNA, unmethylated CpG islands and various other forms of DNA and
RNA classically released by bacteria and viruses.
[0118] The binding of ligand--either in the form of adjuvant used
in vaccinations or in the form of invasive moieties during times of
natural infection--to the TLR marks the key molecular events that
ultimately lead to innate immune responses and the development of
antigen-specific acquired immunity. The very fact that TLR
activation leads to adaptive immune responses to foreign entities
explains why so many adjuvants used today in vaccinations are
developed to mimic TLR ligands. So far, single ligands have been
used as vaccine adjuvants. However, studies suggest that the
combination of more than one adjuvant with either an interferon or
an interleukin could produce a synergistic enhancement of immune
response. Maria Wysocka1, Noor Dawany, Bernice Benoit, Andrew V.
Kossenkov, Andrea B. Troxel, Joel M. Gelfand, Michael kelly Sell1,
Louise C. Showe, & Alain H. Rook (2011). "Synergistic
enhancement of cellular immune responses by the novel Toll receptor
7/8 agonist 3M-007 and interferon-.gamma.: implications for therapy
of cutaneous T-cell lymphoma". Leuk Lymphoma 52 (10): 1970-1979.
PMID 21942329. See also Ghosh T K, Mickelson D J, Fink J, Solberg J
C, Inglefield J R, Hook D, Gupta S K, Gibson S, Alkan S S. (2006).
"Toll-like receptor (TLR) 2-9 agonists-induced cytokines and
chemokines: I. Comparison with T cell receptor-induced responses".
Cellular Immunology 243 (1):
48-57.doi:10.1016/j.cellimm.2006.12.002. PMID 17250816.
[0119] It is believed that upon activation, TLRs recruit adapter
proteins (proteins that mediate other protein-protein interactions)
within the cytosol of the immune cell in order to propagate the
antigen-induced signal transduction pathway. To date, four adapter
proteins have been well-characterized. These proteins are known as
MyD88, Trif, Tram and TIRAP (also called Ma). These recruited
proteins are then responsible for the subsequent activation of
other downstream proteins, including protein kinases (IKKi, IRAK1,
IRAK4, and TBK1) that further amplify the signal and ultimately
lead to the upregulation or suppression of genes that orchestrate
inflammatory responses and other transcriptional events. Some of
these events lead to cytokine production, proliferation, and
survival, while others lead to greater adaptive immunity. The high
sensitivity of TLR for microbial ligands is what makes adjuvants
that mimic TLR ligands such a prime candidate for enhancing the
overall effects of antigen specific vaccinations on immunological
memory.
[0120] Finally, the expression of TLRs is vast as they are found on
the cell membranes of innate immune cells (DCs, macrophages,
natural killer cells), cells of the adaptive immunity (T and B
lymphocytes) and non-immune cells (epithelial and endothelial
cells, fibroblasts). See Yamamoto M, Sato S, Hemmi H, Uematsu S,
Hoshino K, Kaisho T, Takeuchi O, Takeda K, Akira S (2003). "TRAM is
specifically involved in the Toll-like receptor 4-mediated
MyD88-independent signaling pathway". Nat Immunol 4 (11): 1144-50.
doi:10.1038/ni986. PMID 14556004.
[0121] This further substantiates the importance of administering
vaccines with adjuvants in the form of TLR ligands, as they will be
capable of eliciting their positive effects across the entire
spectrum of innate and adaptive immunity. Nevertheless, there are
adjuvants whose immune-stimulatory function completely bypasses the
TLR signaling pathway. While all TLR ligands are adjuvants, not all
adjuvants are TLR ligands.
INVENTION IN GENERAL
[0122] A composition of the invention includes two basic components
which are a pharmaceutically acceptable carrier and a group of
particles in the carrier. Each component, i.e. (a) the carrier and
(b) the particles include an adjuvant and the adjuvants are
different from each other. The inclusion of one type of adjuvant in
the particles and a different adjuvant in the liquid carrier
dramatically improves the response obtained with the
composition.
[0123] The particles are substantially spherical and have a
diameter in a range of 10 to 25 microns with a margin of error of
about .+-.20%. The particles are manufactured in a manner so as to
incorporate within them a CpG oligodeoxynucleotide. The particles
are comprised of a biocompatible polymer and also incorporate an
antigen which may be an amino acid sequence consisting of 8 to 12
amino acids.
[0124] Those skilled in the art will understand that a composition
or formulation of the invention is generally comprised of a carrier
which may be a solid or liquid carrier such as an aqueous carrier
and the carrier may comprise a first adjuvant. The formulation or
composition further comprises a group of particles which may be 10,
100, 500, or 1,000 or more particles. The particles are comprised
of a biocompatible polymer, an adjuvant different from the adjuvant
in the carrier and an antigen. The particles are substantially
spherical and have a diameter in a range of from about 10 microns
to 20 microns.+-.20%. In general the adjuvant present in the
carrier is present in a relatively small amount e.g. 0.05% to 5% by
weight based on the total weight of the composition or formulation.
The first adjuvant might be present in larger amounts such as an
amount within a range of 5 to 50% but more generally would be
present in an amount of about 1% by weight based on the total
weight of the formulation or composition. In a similar manner the
antigen component of the particles as well as the second adjuvant
component of the particles is present in a relatively small amount
such as 0.05% to 5% by weight. Although each component might be
present in larger amounts such as an amount in the range of 5 to
50% by weight they may each be present in an amount of about 1% by
weight.+-.10%.
[0125] The carrier is preferably liquid and aqueous based and may
contain small amounts of components typically included within
pharmaceutical carriers such as buffers, preservative and salts.
The carrier also includes a monophosphoryl lipid A (MPL)
adjuvant.
[0126] Formulations of the invention comprise one group or a
plurality of groups of spherical particles, wherein the particles
in each group may be (a) different or (b) identical in terms of the
antigen and further wherein the particles in different groups may
include a different active ingredient (antigen) from other groups.
The particles are also comprised of one or more adjuvants and the
particles are in a carrier comprised of one or more adjuvents. The
formulation may be designed such that a population of
antigen-presenting cells (APC) can be presented with a plurality of
antigen species, formulated in such a way that any one antigen
presenting cell will take up and present only a limited number of
the antigen species, i.e. less than 5, less than 3, usually only a
single antigen species. There is evidence that presentation of a
plurality of epitopes by a single APC may result in immunodominance
of a single epitope, which may be undesirable in situations where
overall responsiveness to the plurality of epitopes is desirable.
For example, see Rodriguez, et al., "Immunodominance in
Virus-Induced CD8+ T-Cell Responses Is Dramatically Modified by DNA
Immunization and Is Regulated by Gamma Interferon" Journal of
Virology, 76(9):4251-4259 (May 2002) and Yu et al., "Consistent
Patterns in the Development and Immunodominance of Human
Immunodeficiency Virus Type I (HIV-1)-Specific CD8+ T-Cell
Responses following Acute HIV-1 Infection" Journal of Virology,
76(17):8690-9701 (September 2002), both incorporated herein by
reference.
[0127] The invention accomplishes the desired result by placing an
antigen and an adjuvent on or in a particle of a defined size of a
biocompatible polymer, usually in a form that is approved by the
United States Food and Drug Administration for administration to
humans. Vaccine formulations may be comprised of a pharmaceutically
acceptable carrier. The carrier includes an adjuvant and can come
in a variety of forms depending on the mode of administration such
as injection, nasal, inhalation, oral, etc. In addition to the
carrier the formulation includes a plurality of spherical particles
which particles are comprised of: (1) a biocompatible polymer, (2)
an antigen, e.g. a peptide antigen and (3) an adjuvent. The
particles may be generally spherical in shape and have a defined
diameter in a range of 10 microns to 20 microns, or about 10
microns.+-.20%, or .+-.10% or .+-.5% or .+-.2%. The diameter of the
particles may be designed such that an antigen presenting cells
such as dendritic cell can consume only a single particle, and not
consume two or more particles. Each particle may contain a large
number of identical (or in some embodiment different) compounds,
for example peptide species. Thus, each group of particles may
consist essentially only of the biocomparable particle, the
adjuvant and copies of one compound, e.g. peptides which have the
same amino acid sequence. In other embodiments the antigen
containing particles may include more than a single compound or
species, e.g. two, three, four, five or a plurality of compounds,
provided that, in the case of peptides, the different compounds
included do not exhibit immunodominance with respect to each
other.
[0128] The particles from a single group may consist essentially of
a biocomparable polymer, an adjuvant and a single antigen species
that is presented by a class I protein of the APC. Further, the
number of particles within one group may be the same as or may be
different from the number of particles within another group.
[0129] Some examples of biocompatible polymers useful in the
present invention include hydroxyaliphatic carboxylic acids, either
homo- or copolymers, such as poly(lactic acid), poly(glycolic
acid), Poly(dl-lactide/glycolide, poly(ethylene glycol);
polysaccharides, e.g. lectins, glycosaminoglycans, e.g. chitosan;
celluloses, acrylate polymers, and the like. The particle size is
selected to (a) be sufficiently small that it is capable of uptake
and processing by an antigen presenting cell; and (b) be
sufficiently large that an APC will generally not be capable of
taking up more than one particle.
[0130] Each set, or group, of particles comprises a single antigen,
e.g. peptide species, e.g. peptides of identical sequences. The
peptide antigen may be other than a sequence determined to be an
immunodominant sequence.
[0131] Groups of particles may be combined as a composition or
formulation comprising particles with a plurality of peptide
species. For example, two groups of particles provide at least two
peptide species and larger numbers of groups can provide, at least
3, at least 4, at least 5, at least 10, at least 20, and usually
not more than 50, not more than 40, not more than 30 peptide
species via corresponding groups of spheres. In one embodiment, all
of the particles are substantially spherical and have a diameter in
a range of more than 10 microns and less than 20 microns. Each
particle is comprised of a biocompatible polymer an adjuvant and an
antigen.
[0132] In some embodiments the adjuvant and antigen are embedded in
the particle, for example by mixing adjuvant, peptides and polymers
prior to formation of the particles. In other embodiments the
antigens are coupled to the particle surface or the particles are
encapsulated inside an outer spherical shell. The spherical
particle surface may be optionally textured to simulate, to a
degree, the surface of an infectious bacteria, virus or other
pathogen.
[0133] Particles in a formulation may be heterogenous or homogenous
in size, usually homogeneous, where the variability may be not more
than 100% of the diameter, not more 50%, not more than 20%, not
more than 10%, not more than 2%, etc. Particle sizes are may be
about 8 .mu.m in diameter, about 10 .mu.m, about 12 .mu.m about 14
.mu.m, about 15 .mu.m, about 16 .mu.m, about 17, about 18 .mu.m,
about 20 .mu.m, not more than about 25 .mu.m diameter.
[0134] The optimum size for a particular peptide or class of
peptides may be determined empirically by various methods. For
example, two different peptides or adjuvants may be detectably
labeled with two different fluorophores, and used to prepare
particles of the invention. A mixture of the particles is provided
to antigen presenting cells, which are then viewed by optical
microscope, flow cytometry, etc. to determine if a single
fluorophore or if multiple fluorophores are present in any single
APC, where the size of particle that provides for exclusive uptake
is chosen (see FIGS. 1 and 2). Functional tests may also be
performed, e.g. by providing particles with the cognate antigens
for different T cell lines and determining if one or both lines are
activated by an APC.
[0135] In order to determine the precise size which is desirable
for the particles, various types of labeling can be used. In
addition to the fluorophores referred to above, labeling can be
performed with semiconductor nanocrystals which are generally
referred to as quantum dots. The purpose of carrying out the
experiment is to determine a size at which the antigen presenting
cells such as the macrophage can consume only a single particle.
The size would be too large if the macrophage cannot consume the
particle. The size would be too small if the macrophage can consume
more than one particle.
[0136] The optimum size of particle to achieve the desired result
may vary depending on the charge of the peptide that is being
presented, for example a positively charged peptide may be more
readily ingested by an APC than a neutral or negatively charged
peptide. In some embodiments each peptide is individually optimized
for a microsphere size that achieves exclusive uptake, and thus a
formulation of a plurality of microsphere/peptide combinations may
be heterogenous in size, although the size for a peptide species
will be narrowly defined.
[0137] Based on the above it will be understood that one aspect of
the invention is the use of groups of particles of the invention to
determine information about the operation of the immune system,
particularly with respect to antigen presentation. The groups of
particles provide information on how the immune system deals with
different antigens in the particles. Thus, the groups of particles
of the invention can provide valuable information on how an immune
system operates and as such what formulations will provide a
desired immune response.
[0138] In some embodiments of the invention, a formulation is
provided of PLGA microspheres of a defined size from 14 .mu.m to 16
.mu.m in diameter (.+-.20%, .+-.10% m.+-.5%, .+-.2%), where each
microsphere comprises an adjuvant and at least one peptide antigen
species, and where from 10 to 30 different peptide antigen species
are present in the formulation via 10 to 30 different groups of
particles. In some embodiments the peptide antigen is an antigen of
a virus such as hepatitis C virus (HCV) or human immunodeficiency
virus (HIV-1).
Antigen Presenting Cells
[0139] The three major classes of antigen presenting cells are
dendritic cells (DCs), macrophages, and B cells, but dendritic
cells are considerably more potent on a cell-to-cell basis and are
the only antigen presenting cells that activate naive T cells. DC
precursors migrate from bone marrow and circulate in the blood to
specific sites in the body, where they mature. This trafficking is
directed by expression of chemokine receptors and adhesion
molecules. Upon exposure to antigen and activation signals, the DCs
are activated, and leave tissues to migrate via the afferent
lymphatics to the T cell rich paracortex of the draining lymph
nodes. The activated DCs then secrete chemokines and cytokines
involved in T cell homing and activation, and present processed
antigen to T cells. The groups of particles of the invention
provide information on how to best present processed antigens to T
cells to obtain a desired immune response.
[0140] DCs mature by upregulating costimulatory molecules (CD40,
CD80 and CD86), and migrate to T cell areas of organized lymphoid
tissues where they activate naive T cells and induce effector
immune responses. In the absence of such inflammatory or infectious
signals, however, DCs present self-antigens in secondary lymphoid
tissues for the induction and maintenance of self-tolerance.
Dendritic cells include myeloid dendritic cells and plasmacytoid
dendritic cells.
[0141] For purposes of the invention, e.g. determining the uptake
of particles of any formulation including a vaccine formulations by
APC, any one of the classes of APC may be used, including without
limitation immature DC, monocytes, mature myeloid DC, mature pDC,
etc. For example see Foged et al (2005) International Journal of
Pharmaceutics 298(2): 315-322; Reece et al. (2001) Immunology and
Cell Biology 79:255-263; Tel et al. (2010) J. Immunol.
184:4276-4283, each herein specifically incorporated by
reference.
Antigens
[0142] The term "antigen" as used herein includes meanings known in
the art, and means a molecule or portion of a molecule, frequently
for the purposes of the present invention a polypeptide molecule
(amino acid sequence), that can react with a recognition site on an
antibody or T cell receptor. The term "antigen" also includes a
molecule or a portion of a molecule that can, either by itself or
in conjunction with an adjuvant or carrier, elicit an immune
response (also called an "immunogen"). The groups of particles of
the invention provide information on how to best structure antigen
components in the particles to increase or decrease an immune
response in a desired way.
[0143] The "specificity" of an antibody or T cell receptor refers
to the ability of the variable region to bind with high affinity to
an antigen. The portion of the antigen bound by the immune receptor
is referred to as an epitope, and an epitope is that portion of the
antigen which is sufficient for high affinity binding. An
individual antigen typically contains multiple epitopes, although
there are instances in which an antigen contains a single epitope.
In some embodiments of the invention, a plurality of peptide
fragments representing individual epitopes are derived from a
protein antigen. Where the antigen is a peptide, generally a linear
epitope will be at least about 8 amino acids in length, and not
more than about 15 to 22 amino acids in length. A T cell receptor
recognizes a more complex structure than antibodies, and requires
both a major histocompatibility antigen binding pocket and an
antigenic peptide to be present. The binding affinity of T cell
receptors to epitopes is lower than that of antibodies to epitopes,
and will usually be at least about 10.sup.-4 M, more usually at
least about 10.sup.-6 M, 10.sup.-6M, 10.sup.-7M.
[0144] Antigens of interest for the purposes of the invention
include pathogens and portions thereof, e.g. proteins, peptides,
polysaccharides, etc., e.g. virus, bacteria, protozoans, etc.;
tumor antigens, and the like. Viral pathogens of interest include
retroviral pathogens, e.g. HIV-1; HIV-2, HTLV, FIV, SIV, etc.;
influenza, smallpox (vaccinia), measles, mumps, rubella,
poliovirus, rotavirus, varicella (chickenpox), hepatitis A, B, C, D
virus, bacterial antigens, tumor antigens, and the like. Microbes
of interest include, but are not limited to the following, include:
Citrobacter sp.; Enterobacter sp.; Escherichia sp., e.g. E. coli;
Klebsiella sp.; Morganella sp.; Proteus sp.; Providencia sp.;
Salmonella sp., e.g. S. typhi, S. typhimurium; Serratia sp.;
Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp.,
e.g. Y. pestis, Y. pseudotuberculosis, Y enterocolitica;
Franciscella sp.; Pasturella sp.; Vibrio sp., e.g. V. cholerae, V.
parahemolyticus; Campylobacter sp., e.g. C. jejuni; Haemophilus
sp., e.g. H. influenzae, H. ducreyi; Bordetella sp., e.g. B.
pertussis, B. bronchiseptica, B. parapertussis; Brucella sp.,
Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc. Other
bacteria of interest include Legionella sp., e.g. L. pneumophila;
Listeria sp., e.g. L. monocytogenes; Staphylococcus sp., e.g. S.
aureus Mycoplasma sp., e.g. M. hominis, M. pneumoniae;
Mycobacterium sp., e.g. M. tuberculosis, M. leprae; Treponema sp.,
e.g. T. pallidum; Borrelia sp., e.g. B. burgdorferi; Leptospirae
sp.; Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp.,
e.g. C. trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp.,
e.g. H. pylori, etc.
[0145] Antigenic peptides can include purified native peptides,
synthetic peptides, recombinant proteins, crude protein extracts,
attenuated or inactivated viruses, cells, micro-organisms, or
fragments of such peptides. Immunogenic peptides can be native or
synthesized chemically or enzymatically. Any method of chemical
synthesis known in the art is suitable. Solution phase peptide
synthesis can be used to construct peptides of moderate size or,
for the chemical construction of peptides, solid phase synthesis
can be employed. Atherton et al. (1981) Hoppe Seylers Z. Physiol.
Chem. 362:833-839. Proteolytic enzymes can also be utilized to
couple amino acids to produce peptides. Kullmann (1987) Enzymatic
Peptide Synthesis, CRC Press, Inc. Alternatively, the peptide can
be obtained by using the biochemical machinery of a cell, or by
isolation from a biological source. Recombinant DNA techniques can
be employed for the production of peptides. Hames et al. (1987)
Transcription and Translation: A Practical Approach, IRL Press.
Peptides can also be isolated using standard techniques such as
affinity chromatography.
[0146] The groups of particles of the invention can be used to
determine which antigens and antigen synthesis techniques result in
obtaining an immune response desired.
Type of Vaccines
[0147] The groups of particles of the invention can use epitope
portions of vaccine materials as described below to determine which
positions of these materials obtain the desired immune response.
Such information can be used to create safer, less expensive and
more effective vaccines.
[0148] Some vaccines contain inactivated, but previously virulent,
micro-organisms that have been destroyed, i.e. rendered inactive
such as with chemicals or heat. Examples are the influenza vaccine,
cholera vaccine, bubonic plague vaccine, polio vaccine, hepatitis A
vaccine, and rabies vaccine. Other vaccines contain live,
attenuated microorganisms. Many of these are live viruses that have
been cultivated under conditions that disable their virulent
properties, or which use closely-related but less dangerous
organisms to produce a broad immune response, however some are
bacterial in nature. They typically provoke more durable
immunological responses and are the preferred type for healthy
adults. Examples include the viral diseases yellow fever, measles,
rubella, and mumps and the bacterial disease typhoid. The live
Mycobacterium tuberculosis vaccine developed by Calmette and Guerin
is not made of a contagious strain, but contains a related species
called "BCG".
[0149] Toxoid vaccines are made from inactivated toxic compounds
that cause illness rather than the micro-organism. Examples of
toxoid-based vaccines include tetanus and diphtheria. Toxoid
vaccines are known for their efficacy. Not all toxoids are for
micro-organisms; for example, Crotalus atrox toxoid is used to
vaccinate dogs against rattlesnake bites.
[0150] In subunit vaccine, a fragment of a microorganism is
utilized to induce an immune response. Examples include the subunit
vaccine against Hepatitis B virus, which is composed of only the
surface proteins of the virus (previously extracted from the blood
serum of chronically infected patients, but now produced by
recombination of the viral genes into yeast); the virus-like
particle (VLP) vaccine against human papillomavirus (HPV) that is
composed of the viral major capsid protein, and the hemagglutinin
and neuraminidase subunits of the influenza virus.
[0151] In conjugate vaccines, the polysaccharides present on
bacterial outer coats are conjugated to a carrier to induce a more
robust response, e.g. in the Haemophilus influenzae type B
vaccine.
[0152] Vaccines currently in development include dendritic cell
vaccines, which combine dendritic cells with antigens, which have
shown some positive preliminary results for treating brain tumors.
In recombinant vector vaccines, the physiology of one
micro-organism is combined with the DNA of the other, to create
immunity against diseases that have complex infection processes.
DNA vaccination, created from an infectious agent's DNA, has been
developed. It works by insertion (and expression, triggering immune
system recognition) of viral or bacterial DNA into human or animal
cells. Some cells of the immune system that recognize the proteins
expressed will mount an attack against these proteins and cells
expressing them. Because these cells live for a very long time, if
the pathogen that normally expresses these proteins is encountered
at a later time, they will be attacked instantly by the immune
system. One advantage of DNA vaccines is that they are very easy to
produce and store. T-cell receptor peptide vaccines are under
development for several diseases using models of Valley Fever,
stomatitis, and atopic dermatitis. These peptides have been shown
to modulate cytokine production and improve cell mediated immunity.
Other vaccines are in development to target bacterial proteins that
are involved in complement inhibition, which would neutralize a key
bacterial virulence mechanism.
[0153] While most vaccines are created using inactivated or
attenuated compounds from micro-organisms, synthetic vaccines are
composed mainly or wholly of synthetic peptides, carbohydrates or
antigens.
[0154] Vaccines may be monovalent (also called univalent) or
multivalent (also called polyvalent). A monovalent vaccine is
designed to immunize against a single antigen or single
microorganism. A multivalent or polyvalent vaccine is designed to
immunize against two or more strains of the same microorganism, or
against two or more microorganisms. In certain cases a monovalent
vaccine may be preferable for rapidly developing a strong immune
response.
[0155] In order to provide the best protection against an
infection, children are recommended to receive vaccinations as soon
as their immune systems are sufficiently developed to respond to
particular vaccines, with additional "booster" shots often required
to achieve "full immunity". This has led to the development of
complex vaccination schedules. In the United States, the Advisory
Committee on Immunization Practices, which recommends schedule
additions for the Centers for Disease Control and Prevention,
recommends routine vaccination of children against: hepatitis A,
hepatitis B, polio, mumps, measles, rubella, diphtheria, pertussis,
tetanus, HiB, chickenpox, rotavirus, influenza, meningococcal
disease and pneumonia. The large number of vaccines and boosters
recommended (up to 24 injections by age two) has led to problems
with achieving full compliance. In order to combat declining
compliance rates, various notification systems have been instituted
and a number of combination injections are now marketed (e.g.,
Pneumococcal conjugate vaccine and MMRV vaccine), which provide
protection against multiple diseases.
[0156] Besides recommendations for infant vaccinations and
boosters, many specific vaccines are recommended at other ages or
for repeated injections throughout life--most commonly for measles,
tetanus, influenza, and pneumonia. Pregnant women are often
screened for continued resistance to rubella. The human
papillomavirus vaccine is currently recommended in the U.S. and UK
for ages 11-25. Vaccine recommendations for the elderly concentrate
on pneumonia and influenza, which are more deadly to that group. In
2006, a vaccine was introduced against shingles, a disease caused
by the chickenpox virus, which usually affects the elderly.
[0157] Use of groups of particles of the invention can provide
information that will allow for the reformulation of current
vaccines, e.g. reducing undesired immune responses via small
antigen components, improving patient compliance by obtaining an
immune response that does not require a follow-up booster.
Information obtained via the groups of particles of the invention
can also be combined with patient specific HLA information to
tailor immunogens to specific groups of HLA types.
Tattoo Ink
[0158] A vaccine formulation of the invention may comprise ink to
mark the skin of the human patient to which the vaccine is
administered. The ink may be conventional ink used in tattooing, or
may be ink that is only visible under a specific type of light such
as laser light, or a black light that emits ultraviolet radiation.
The ink and vaccine may be administered in a single formulation or
in two separate formulations. The ink may be free in the
formulation or may be encapsulated in microparticles of similar
size to vaccine particles of the invention. The ink may be
encapsulated by a process as disclosed within U.S. Pat. No.
6,116,516 issued Sep. 12, 2000 to Alfonso Ganan-Calvo, herein
incorporated by reference. The ink particles may be smaller than
any vaccine particle because the immunodominance problem is not an
issue with ink. The small encapsulate ink particles may enhance the
immune response to be generated in response to the antigens of the
formulation. Further, enhancement of that immune response may be
generated by using a tattoo needle administering device to
administer the formulation.
General Overview
[0159] Vaccine compositions of the invention may be formulated for
delivery to a patient via a tattoo needle or similar device. Such
vaccine compositions may include a tattoo ink that can be used to
create an identifying marking on a patient for purposes of, e.g.,
identifying the person, the date of administration and/or the
particular vaccine composition that was administered to the
patient. In some embodiments, tattoo ink compositions are provided
wherein the tattoo ink does not comprise a vaccine, and is simply
used to create an identifying marking on the patient's skin.
[0160] Suitable tattoo inks for use with the compositions and
methods of the present disclosure include tattoo inks that are
visible to the naked eye, as well as tattoo inks that are invisible
to the naked eye, but which may be selectively activated for
identification purposes. Other suitable tattoo inks include
erasable or removable tattoo inks that can be removed when no
longer needed, and tattoo ink compositions that are not permanent,
such as those that are designed to fade away and disappear with
time. Exemplary tattoo ink compositions are provided below.
Traditional Tattoo Ink Compositions
[0161] In some embodiments of the present disclosure, tattoo ink
compositions may comprise traditional tattoo pigments and dyes,
such as, e.g., India ink. Traditional tattoo inks are generally
visible with the naked eye and are commercially available from a
variety of sources.
Semi-Permanent Tattoo Ink Compositions
[0162] In some embodiments of the present disclosure, tattoo ink
compositions are provided which remain in the dermis for a
predetermined period of time (e.g., 3, 6, 9, months or 1, 2, 5, 10
years, etc.) and then spontaneously disappear. These
"semi-permanent" or "temporary" tattoo inks are produced by
entrapping, encasing, complexing, incorporating, or encapsulating
appropriate pigments (pigments which are readily eliminated when
present by themselves in the dermis) into tattoo pigment vehicles
which slowly bioabsorb, bioerode, or biodegrade over a
predetermined period of time, such as at a constant rate slowly
over a five-year period, or can release the pigments over a short
period of time once a specific percentage of the tattoo pigment
vehicle has been absorbed. For example, all of the pigment may be
released between the fourth and fifth years.
[0163] One example of a tattoo pigment vehicle which releases the
pigment continuously over a predetermined period is one in which
the pigment is incorporated or mixed in throughout the entire
substance of the tattoo pigment vehicle to form color-carrying
particles. When these pigment/vehicle complexes are introduced into
the dermis (in the form of a tattoo), the tattoo pigment vehicle
slowly bioabsorbs, releasing the pigment from the dissolving tattoo
pigment vehicle maturely, eliminating the pigment from the dermis.
When all of the pigment/vehicle complexes have been absorbed, the
tattoo is no longer visible.
[0164] To release the pigment over a short period of time,
bioabsorbable microcapsules or microfiakes are used as the tattoo
pigment vehicle. With microcapsules, pigment/vehicle complexes
comprise a core of pigment surrounded by the tattoo pigment
vehicle, which maintains its integrity until a certain threshold
percentage of the tattoo pigment vehicle is dissolved, bioeroded,
or bioabsorbed. At this point, the tattoo pigment vehicle no longer
protects the pigment from elimination. The pigment is released into
the dermis, where it is eliminated over a relatively short period
of time.
[0165] Alternatively, microfiakes made of pigment and tattoo
pigment vehicle, in which the pigment is mixed throughout the
microfiakes, maintain a relatively consistent pigmented surface
area during the process of bioabsorption. Over a predetermined
period of time, the visible pigmented surface dissolves, similar to
the melting of a frozen lake or pond.
[0166] The tattoo pigment vehicle for the pigment or dye comprises
any biologically tolerated material that retains the pigment or dye
in the dermis, for whatever time or under whatever conditions are
desired. In any of these cases, the tattoo pigment vehicle carries
a colored pigment or dye which can be administered into the dermis
in any pattern or configuration in a manner similar to conventional
tattooing. The tattoo pigment vehicle is sufficiently transparent
or translucent so as to permit the color of the pigment or dye to
show through and be visible. Preferably, the pigment or dye
comprises particles smaller than 1 micron.
[0167] For producing semi-permanent tattoos, the pigments or dyes
are entrapped, encased, complexed, incorporated, encapsulated, or
otherwise associated in or with tattoo pigment vehicles composed of
bioabsorbable, bioerodable, or biodegradable material. The material
is designed to bioabsorb, bioerode, or biodegrade over a
predetermined period of time so that the tattoo ink, when
administered into the dermis, creates a tattoo which lasts only
until the tattoo pigment vehicle bioabsorbs. Upon partial or
complete bioabsorption of the tattoo pigment vehicle, the pigment
or dye is released, allowing its elimination from the dermis.
[0168] A great many biodegradable polymers exist, and the length of
time which the tattoo lasts in a visible state in the dermis is
determined by controlling the type of material and composition of
the tattoo pigment vehicle. Among the bioabsorbable, bioerodable,
or biodegradable polymers that can be used are those disclosed in
Higuchi et al., U.S. Pat. Nos. 3,981,303, 3,986,510, and 3,995,635,
including zinc alginate poly(lactic acid), poly(vinyl alcohol),
polyanhydrides, and poly(glycolic acid). Alternatively, microporous
polymers are suitable, including those disclosed in Wong, U.S. Pat.
No. 4,853,224, such as polyesters and polyethers, and Kaufman, U.S.
Pat. Nos. 4,765,846 and 4,882,150.
[0169] Other polymers which degrade slowly in vivo are disclosed in
Davis et al., U.S. Pat. No. 5,384,333, which are biodegradable
polymers which are solid at 20-37.degree. C. and are flowable,
e.g., a liquid, in the temperature range of 38-52.degree. C. In
preparing a semi-permanent tattoo, the dye or pigment is
incorporated in the polymer matrix, and the system can be warmed to
approximately 50.degree. C., where it liquefies. The system is then
injected into the dermis in a desired tattoo design, where it cools
and re-solidifies.
[0170] For this type of semi-permanent tattoo pigment vehicle, any
biodegradable polymer system which has the following
characteristics can be used, including homopolymers, copolymers,
block copolymers, waxes and gels, as well as mixtures thereof. A
preferred polymer system is a triblock copolymer of the general
formula: A-B-A, where A represents a hydrophobic polymer block, and
B represents a hydrophilic polymer. The monomers and polymers are
preferably linked through ester groups. Preferred hydrophobic
polymers and oligomers include, but are not limited to, units
selected from polyglycolic acid, polyethylene terephthalate,
polybutyl lactone, polycaprolactone, D-polylactic acid,
polytetrafluoroethylene, polyolefins, polyethylene oxide,
polylactic acid, polyglutamic acid, polyL-lysine, and
poly-L-aspartic acid. Preferred hydrophilic polymers include
polyethylene glycol, polypropylene glycol, and poly(vinyl
alcohol).
[0171] Hydrogel matrices or tattoo pigment vehicles for preparing
semi-permanent tattooing inks are formed by crosslinking a
polysaccharide or a mucopolysaccharide with a protein and loading
the dye or pigment into the hydrogel matrices. Proteins include
both full-length proteins and polypeptide fragments, which in
either case may be native, recombinantly produced, or chemically
synthesized. Polysaccharides include both polysaccharides and
mucopolysaccharides.
[0172] A hydrogel in which the pigment or dye can be incorporated
into a tattoo ink is disclosed in Feijen, U.S. Pat. No. 5,041,292.
This hydrogel comprises a protein, a polysaccharide, and a
cross-linking agent providing network linkages therebetween wherein
the weight ratio of polysaccharide to protein in the matrix is in
the range of about 10:90 to about 90:10. The pigment or dye is
mixed into this matrix in an amount sufficient to provide color
when the hydrogel matrix is administered to the dermis.
[0173] Examples of suitable polysaccharides include heparin,
fractionated heparins, heparan, heparan sulfate, chondroitin
sulfate, and dextran, including compounds described in U.S. Pat.
No. 4,060,081 to Yannas et al. Using heparin or heparin analogs
results in reduced immunogenicity in terms of a response to the
heparin. The protein component of the hydrogel may be either a
full-length protein or a polypeptide fragment. The protein may be
in native form, recombinantly produced, or chemically synthesized.
The protein composition may also be a mixture of full-length
proteins and/or fragments. Typically, the protein is selected from
the group consisting of albumin, casein, fibrinogen,
gamma-globulin, hemoglobin, ferritin and elastin. The protein
component may also be a synthetic polypeptide, such as poly
(a-amino acid), polyaspartic acid or polyglutamic acid. Albumin is
the preferred protein component of the matrix, as it is an
endogenous material which is biodegradable in blood and tissue by
proteolytic enzymes. Furthermore, albumin prevents adhesion of
thrombocytes and is nontoxic and nonpyrogenic.
[0174] In forming hydrogels containing pigments or dyes; the
polysaccharide or mucopolysaccharide and the protein are dissolved
in an aqueous medium, followed by addition of an amide bond-forming
cross-linking agent. A preferred crosslinking agent for this
process is a carbodiimide, preferably the water-soluble diimide
N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide. In this method, the
cross-linking agent is added to an aqueous solution of the
polysaccharide and protein at an acidic pH and a temperature of
about 0 to 50.degree. C., preferably from about 4 to about
37.degree. C., and allowed to react for up to about 48 hours. The
hydrogel so formed is then isolated, typically by centrifugation,
and washed with a suitable solvent to remove uncoupled
material.
[0175] Alternatively, a mixture of the selected polysaccharide or
mucopolysaccharide and protein is treated with a crosslinking agent
having at least two aldehyde groups to form Schiff-base bonds
between the components. These bonds are then reduced with an
appropriate reducing agent to give stable carbon-nitrogen
bonds.
[0176] Once the hydrogel is formed, it is loaded with the pigment
or dye by immersing the hydrogel in a solution or dispersion of the
pigments or dye. The solvent is then evaporated. After
equilibration, the loaded hydrogels are dried in vacuo under
ambient conditions and stored.
[0177] Virtually any pigment or dye may be loaded into the hydrogel
vehicles, providing that surface considerations, such as surface
charge, size, geometry and hydrophilicity, are taken into account.
For example, incorporation and release of a high-molecular weight
dye will typically require a hydrogel having a generally lower
degree of cross-linking. The release of a charged pigment or dye
will be strongly influenced by the charge and charge density
available in the hydrogel, as well as by the ionic strength of the
surrounding media.
[0178] The rate of pigment or dye release from the vehicles can
also be influenced by post-treatment of the hydrogel formulations.
For example, heparin concentration at the hydrogel surface can be
increased by reaction of the formulated hydrogels with activated
heparin (i.e., heparin reacted with carbonyldiimidazole and
saccharine) or with heparin containing one aldehyde group per
molecule. A high concentration of heparin at the hydrogel surface
will form an extra "barrier" for positively charged dyes or
pigments at physiological pH values. Another way of accomplishing
the same result is to treat the hydrogels with positively charged
macromolecular compounds like protamine sulfate, polylysine, or
like polymers. Another way of varying hydrogel permeability is to
treat the surfaces with biodegradable block copolymers containing
both hydrophilic and hydrophobic blocks. The hydrophilic block can
be a positively charged polymer, like polylysine, while the
hydrophilic block can be a biodegradable poly(a-amino acid), such
as poly(L-alanine), poly(L-leucine), or similar polymers.
[0179] Another slow-release system used as a tattoo pigment vehicle
for pigments or dyes to form a semi-permanent tattoo is a dye or
pigment and an enzyme encapsulated within a microcapsule having a
core formed of a polymer which is specifically degraded by the
enzyme and a rate controlling skin. The integrity of the shell is
lost when the core is degraded, causing a sudden release of pigment
or dye from the capsule. In this type of system, the microcapsule
consists of a core made up of a polymer around which there is an
ionically-bound skin or shell. The integrity of the skin or shell
depends on the structure of the core. An enzyme is encapsulated
with the biologically-active substance to be released during
manufacture of the core of the microcapsule. The enzyme is selected
to degrade the core to a point at which the core can no longer
maintain the integrity of the skin, so that the capsule falls
apart. An example of such as system consists of an ionically
cross-linked polysaccharide, calcium alginate, which is ionically
coated with a polycationic skin of poly-L-lysine. The enzyme used
to degrade the calcium-alginate coated with poly-L-lysine
microcapsules is an alginase from the bacteria Beneckea pelagio or
Pseudomonas putida. Enzymes exist that degrade most
naturally-occurring polymers. For example, the capsule core may be
formed of chitin for degradation with chitinase.
[0180] Other natural or synthetic polymers may also be used and
degraded with the appropriate enzyme, usually a hydrogenase.
[0181] A particularly preferred bioabsorbable polymer vehicle is a
triblock copolymer of poly caprolactone-polyethylene glycol-poly
caprolactone. This polymer contains ester bonds which hydrolyze in
a hydrophilic environment. The biodegradable polymer matrix should
comprise about 30-99% of the tattoo ink.
[0182] Several mechanisms are involved in the rate and extent of
dye or pigment release. In the case of very high molecular weight
pigments, the rate of release is more depending on the rate of
tattoo pigment vehicle bioabsorption. With lower molecular weight
pigments, the rate of pigment release is more dominated by
diffusion. In either case, depending on the tattoo pigment vehicle
composition selected, ionic exchange can also play a major role in
the overall release profile.
Selectively Activatable Tattoo Ink Compositions
e.g., pH Sensitive
[0183] Formulations of the invention may comprise a selectively
activatable tattoo ink composition and system, wherein the tattoo
ink is changed in color and even appears to disappear by becoming
colorless, with the process being reversible and repeatable. The
compositions and systems disclosed herein are particularly well
suited for use with a pH-sensitive tattoo ink composition, and will
be described below. The compositions and systems of the present
disclosure are equally well suited for use with other types of
chemical sensitivity, such as salinity, glucose levels, etc.
[0184] In one embodiment, the present invention provides a type of
tattoo ink that is visible or invisible under ambient light until
an activating chemical is applied to it. This chemically reactive
adjustable tattoo ink creates a reversible chemical change in the
ink which results in the ink changing color, becoming visible or
invisible. The tattoo is formed by injection of the selectively
alterable intermittent tattoo ink (reactive pigment), along with an
inert carrier and a temporary activating agent (so the ink will at
least be temporarily visible during application), into the skin
with a traditional tattoo delivery system. The selectively
alterable intermittent tattoo ink can then be altered (invisible
ink activated, visible ink deactivated, etc.) such as by the
application of an activating agent either to the skin over the
tattoo site or by a normal or abnormal physiological chemical
change within the body. The activating agent is removed from the
skin by washing, by the application of a neutralizing agent, or by
the natural metabolic functions of the body resulting in the tattoo
being invisible or being returned to its deactivated state.
[0185] The selectively alterable intermittent tattoo ink of the
present disclosure may be applied as any other tattoo ink, and will
provide the wearer with a permanent tattoo that will be visible
only if and when the wearer wishes, and the colors of which are
adjustable and reversible.
[0186] In one embodiment, the tattoo ink system comprises pH
sensitive pigments, a harmless non-pigmented carrier, an activating
agent (such as an ointment which may be applied with a
pre-moistened disposable towelette wipe, disclosed in U.S. Pat. No.
6,287,582, or a normal or abnormal physiological change), and a
neutralizing agent (such as an ointment, spray-on mist, or
pre-moistened disposable towelette wipe). Both the activating agent
and neutralizing agent can be applied with a towelette, spray-on
mist, or ointment. The pigment in the ink can be activated in a
number of ways; by application of a solution of specific pH value
such as by way of a pre-moistened wipe, spray-on mist, or
application of an ointment of specific pH to the tattoo location in
which the H+ ions will diffuse into or out of the skin by way of
osmosis activating the pigments; by a normal physiological change
in pH created by heavy exercise as in exercise-induced acidosis; or
by a pathological acidosis such as metabolic acidosis.
[0187] Non-limiting examples of suitable pH-sensitive pigments
include: bromocresol purple, chlorophenol red, bromophenol blue,
p-nitrophenol, phenol red, neutral red, rosolic red, cresol red,
thymol blue, and phenolpthalein. These chemicals are reagents that,
when exposed to changing pH levels, react by changing color. By
combining the above chemicals, tattoo ink compositions can be
created that are invisible, change color, change shape, change
size, or disappear when exposed to the appropriate pH.
[0188] While only blood pH values ranging from 6.8 to 7.8 are
compatible with life, the physiological pH of the skin typically
ranges from 5.0 to 9.0. Commercial skin products are known and
available on the market boasting as being noncorrosive even at pH
levels as low as 1.0 (the exemplary pigments listed above all react
within a pH range from 5.0 to 9.0.) In general, the compositions of
the present disclosure have pigments that are capable of reacting
over a pH value of between 4.4 and 10.5.
[0189] By preparing a tattoo ink which utilizes a pH sensitive
pigment, such as those previously described, or the indicators
listed in Table 1, a chemically adjustable intermittent tattoo ink
can be made, wherein the tattoo ink changes in color such that the
tattoo can change in appearance, color and even vanish. This color
change process is fully reversible.
[0190] A suitable carrier of the pH sensitive pigment chemicals can
be glycerin, ethyl alcohol, purified water, witch hazel, as well as
any other existing carriers that are in use in the tattoo industry
and are known to those skilled in the art. The tattoo ink is
generally prepared at a pH level necessary for it to be seen in its
activated color so it may be accurately drawn with. The buffering
system of the human body brings the pH of the injection site
(tattoo needle sight) to within the normal physiological
intracellular range of pH from 6.8 to 7.4 at which time the tattoo
would change to its deactivated state.
[0191] One embodiment of an activating agent for the tattoo ink of
the present disclosure is made with Beckman Hydroxide pH Buffer at
pH 12.45 and distilled water. Other embodiments include: sodium
phosphate dibasic, sodium hydroxide, and distilled water; and an
organic acid (such as citric acid or ascorbic acid) and distilled
water. By combining organic acid, sodium phosphate dibasic, and
sodium hydroxide, ointments of various pH levels are produced that
activate the pH-sensitive tattoo inks individually or together. A
further embodiment of the activating agent is a solution of Beckman
Hydroxide Buffer at pH 12.45 being delivered to the tattoo site via
use of an individual pre-moistened towelette wipe, spray-on mist,
or ointment.
[0192] Yet another embodiment of the neutralizing agent for the
selectively alterable intermittent tattoo ink is a solution of an
organic acid and distilled water with a pH of 1 being delivered to
the tattoo site by way of an individual pre-moistened wipe or
towelette, spray-on mist, lotion or ointment.
[0193] Prepared pH ointment such as one consisting of an organic
acid, sodium phosphate dibasic, and sodium hydroxide or Beckman
Hydroxide Buffer at pH 12.45, can be applied topically to the skin
over the tattoo to activate it. Pre-packed individual pre-moistened
wipes or towelettes consisting of solutions of an organic acid,
sodium phosphate dibasic and sodium hydroxide of various pH values
or Beckman Hydroxide Buffer at pH 12.45, as well as a spray-on mist
consisting of the same pH values can be applied to activate the
tattoo ink. A prepared neutralizing ointment such as one consisting
of an organic acid and water at pH 1 in a carrier base of
Ultra-Myossage, or an organic acid and water delivered in a
pre-moistened towelette or spray-on mist can be applied to
deactivate the tattoo. Osmosis drives the H+ ions into the skin,
activating the pH-sensitive pigments or, in the case with some of
the pH-sensitive pigments listed above (or are listed in Table 1)
which need a more basic environment to react, drive the H+ ions out
of the skin, also activating the tattoo ink.
[0194] Referring to Table 1 there are listed various pH indicators,
including descriptions of their color change, pH ranges and
solubility/preparation. The preparation is not to be considered
indicative of the actual ink formulation.
TABLE-US-00001 TABLE 1 Exemplary pH-sensitive tattoo ink
compositions. Tattoo Ink pH Color Composition Change Change
Preparation Quinaldine Red 1.0-2.2 Colorless to 1% in EtOH red
2,4-Dinitrophenol 2.8-4.0 Colorless to Saturated Water yellow
Solution Ethyl Red 4.0-5.8 Colorless to 0.1 g in 50 ml red MEOH +
50 ml water p-Nitrophenol 5.4-6.6 Colorless to 0.1% in water yellow
o-Cresolpthalein 8.2-9.8 Colorless to 0.04% in EtOH red
Phenolphthalein 8.2-10.0 Colorless to 0.05 g in 50 ml pink EtOH +
50 ml water Ethyl-bis (2,4- 8.4-9.6 Colorless to Saturated solution
in dimethylphenyl)acetate blue 50% acetone Thymolphthalein 8.8-10.5
Colorless to 0.04 g in 50 ml blue EtOH + 50 ml water
2,4,6-Trinitrotoluene 11.5-13.0 Colorless to 0.1-0.5% in EtOH
orange 1,3,5-Trinitrobenzene 12.0-14.0 Colorless to 0.1-0.5% in
EtOH orange
[0195] In the following exemplary formulae, percentages are by
weight. Powdered pigments are pH active reagents selected from
Table 1. An exemplary embodiment of the selectively alterable
intermittent tattoo ink formulation is comprised of a mixture of
glycerin, witch hazel, powered pigment and ethyl alcohol in the
range from approximately 64% glycerin, 10% witch hazel, 26%
powdered pigment, and no ethyl alcohol to: 52% glycerin, 23% witch
hazel 17% powdered pigment, 8% ethyl alcohol.
[0196] The following formulations are alternative embodiments that
are equally well suited and provide a suitable alternative for the
commercial products Beckman's 12.45 pH buffer and Ultra-myosage
lotion described herein.
[0197] An exemplary formulation of a corresponding acidic activator
comprises approximately 90% distilled water and 10% citric acid by
weight, which has a pH of approximately 1. This formulation is used
in a spray-on mist and pre-moistened towelletes. An exemplary
acidic lotion formation comprises approximately 48% glycerin, 34%
citric acid, and 18% distilled water. This exemplary lotion
formulation has a pH of approximately 3, as the glycerin acts as a
buffer. Lower pH levels can be provided by using a formulation
containing no or less glycerin.
[0198] An exemplary formulation of a corresponding alkaline
activator is 89% distilled water, 9% sodium phosphate dibasic, and
2% sodium hydroxide, which has a pH of approximately 12.5. An
exemplary alkaline lotion formulation comprises approximately 69%
glycerin, 17% distilled water, and 14% sodium hydroxide, which has
a pH of approximately 12.5.
[0199] The tattoo ink compositions that are either invisible
(clear) or too light to clearly see while deactivated require that
they be temporarily activated for the tattoo artist to use.
Examples of such inks include those with pigments based on:
Thymolphthalein, Phenolphthalein, o-Cresolphthalein, as well as
other pigments. In these cases, the tattoo ink formulations are
temporarily activated such as with the addition of approximately
0.03 grams of solution of 6% sodium hydroxide and 94% distilled
water to 10 grams of ink. The solution temporarily raises the ink
pH level to approximately 10. The temporarily activated ink is
neutralized/deactivated by the body's natural buffering system and
renders the tattoo invisible over time but is visible when the
tattoo is being applied to the patient's skin.
[0200] In some tattoo ink formulations of the present disclosure,
the acidic activator acts as a neutralizer for the inks activated
on the alkaline side of the pH scale and vice a versa. Numerous pH
activated powdered pigments can be made into selectively alterable
intermittent tattoo inks using the above formulations and are
activated by either the alkaline or acidic spray-on mist,
pre-moistened towelletes, or lotions. In general the term lotion is
used to describe a formulation which can be poured while an
ointment does not pour. However, the configuration of a formulation
as either a lotion or an ointment is primarily for user
convenience.
[0201] Numerous modifications and alternative embodiments of the
disclose tattoo ink compositions will be apparent to those skilled
in the art in view of the foregoing description. For example, the
pigments can be further reactive to temperature as well as other
physiological changes and chemical concentrations in the skin.
Furthermore, the activating formulations can be scented with
specific scents, etc. Accordingly, this description is to be
construed as illustrative only and is for the purpose of teaching
those skilled in the art the best mode of carrying out the
invention. Details of the structure may be varied substantially
without departing from the spirit of the invention and the
exclusive use of all modifications, which come within the scope of
the appended claims, is reserved.
UV Visible Tattoo Ink
[0202] The present disclosure generally provides tattoo ink
compositions that are invisible to the naked eye until the tattoo
is exposed to sunlight or another source of ultraviolet radiation,
at which time the tattoo undergoes a photochemical change from
clear or colorless to intensely colored, thereby producing a
visible tattoo. Upon removal from exposure to UV, the tattoo again
becomes invisible. By appropriate formulation, the tattoo can be
made to return to clear and therefore become invisible almost
immediately, or following a predetermined desired period of
time.
[0203] The tattoo ink of the present disclosure is generally
created by mixing a photochromic compound with a suitable carrier,
such as the ink disclosed in U.S. Pat. No. 5,878,155, for example.
The photochromic compound is selected from the spiropyrans and
spiroxazine groups of the photochromic family of compounds, such as
those disclosed in U.S. Pat. No. 5,581,090, for example. The
spiropyrans and spiroxazine compounds normally appear colorless but
undergo photochemical transformation and exhibit intense colors
when exposed to UV radiation.
[0204] The spiropyrans or spiroxazine compounds may be added to the
carrier in a proportion of about 0.1 to about 1.0 percent by
weight. Higher concentrations yield more intense colors. Different
color hues are obtained by mixing two or more spiropyrans or
spiroxazine compounds in the carrier, as necessary or desired.
[0205] During application of the tattoo, it is necessary to expose
the area being tattooed to UV radiation so that the design is
visible to the tattooist. Upon exposure to UV radiation from a lamp
or sunlight, for example, the tattoo becomes fully visible after
only about five (5) seconds.
[0206] A commercially-available stabilizer may be added to the
composition to reduce oxidation processes and thereby extend the
useful life of the photochromic compound. Commercially-available
stabilizers suitable for their performance under sunlight are
Tinuvin 765 and Tinuvin 144 Hindred Amine Light Stabilizers,
available from, e.g., the Additive Division of Ciba Geigy. The
stabilizers may be added in a proportion of from about 2.5% up to
about 3.0% by weight.
[0207] The photochromatic tattoo ink used with formulations of the
invention can be applied using conventional tattooing methods and
equipment, and when visible appears as a conventional tattoo. It
can have the permanence, individuality, artistry and intricate
detail of a conventional tattoo, but remains invisible until
exposed to UV radiation. Thus, the person wearing the tattoo can
dress in formal clothing or swim wear, etc., without displaying the
tattoo, and when desired can activate the tattoo for display simply
by exposing it to UV radiation. If desired, the tattoo can even be
made to remain invisible when the person wearing the tattoo is
exposed to sunlight or other source of UV radiation by covering the
tattoo with an appropriate sun (UV) block.
Producing Particles of the Formulation
[0208] Particles of the formulation can be produced in a number of
different ways.
[0209] However, it is desirable that the particles be produced with
certain specific characteristics. For example, it is desirable that
the particles in a formulation all have the same size.+-.20%,
.+-.10%, .+-.5%, .+-.2%. It is also desirable that the process for
producing the particles produce the particles without damaging
either the adjuvants or the peptides of epitopes on the attenuated
viruses in the particles. Such particles can be produced using a
process referred to as "Flow Focusing" as disclosed within U.S.
Pat. No. 6,116,516 issued Sep. 12, 2000 to Alfonso Ganan-Calvo,
herein incorporated by reference.
[0210] Substantially any biocompatible polymer can be used in
forming the particles. Those skilled in the art will appreciate
that polymers can be selected from the group consisting of
poly(lactic-co-glycolic acid) (PLGA), polycaprolactone,
polyglycolide, polylactic acid, poly-3-hydroxybutyrate. Different
polymer and different degree of polymerization and cross-linking
can be used to obtain a desired release profile of the particles
contents.
[0211] With respect to the peptide, the amino acid sequence in any
given group of particles will be identical and will be chosen based
on the particular pathogen of interest. Identical peptide sequences
are produced and mixed with the desired polymer such as PLGA and
the polymer with the peptides is extruded from a device (as
disclosed within U.S. Pat. No. 6,116,516 which is incorporated
herein by reference in its entirety) to produce particles.
[0212] In the present invention the epitope may consist only of an
amino acid sequence, which could be produced from a virus. Viruses
are grown either on primary cells such as chicken eggs (e.g., for
influenza), or on continuous cell lines such as cultured human
cells (e.g., for hepatitis A). Bacteria are grown in bioreactors
(e.g., Haemophilus influenzae type b). Alternatively, a recombinant
protein derived from the viruses or bacteria can be generated in
yeast, bacteria, or cell cultures. After the antigen is generated,
it is isolated from the cells used to generate it. A virus may need
to be inactivated, possibly with no further purification required.
Recombinant proteins need many operations involving ultrafiltration
and column chromatography.
[0213] The vaccine may be formulated by adding adjuvant,
stabilizers, and preservatives as needed to water, saline solution
or other flowable liquid, all with or without ink which may be ink
visible only under a black light. The adjuvant enhances the immune
response of the antigen, stabilizers increase the storage life, and
preservatives allow the use of multidose vials. Combination
vaccines are harder to develop and produce, because of potential
incompatibilities and interactions among the antigens and other
ingredients involved.
[0214] Vaccine production techniques are evolving. Cultured
mammalian cells are expected to become increasingly important,
compared to conventional options such as chicken eggs, due to
greater productivity and few problems with contamination.
Recombination technology that produces genetically detoxified
vaccine is expected to grow in popularity for the production of
bacterial vaccines that use toxoids. Combination vaccines are
expected to reduce the quantities of antigens they contain, and
thereby decrease undesirable interactions, by using
pathogen-associated molecular patterns. Groups of particles of the
invention can be used to provide combination vaccines.
FORMULATIONS
[0215] The compositions of the invention are especially useful for
administration to an individual in need of immune stimulation (in
the context of, for example, infectious disease, cancer, and
allergy), and generally comprise a plurality of groups of
microspheres of defined size comprising distinct antigen species as
described herein, in a sufficient amount to modulate an immune
response.
[0216] The compositions of the invention optionally comprise a
pharmaceutically acceptable excipient, and may be in various
formulations. As is well known in the art, a pharmaceutically
acceptable excipient is a relatively inert substance that
facilitates administration of a pharmacologically effective
substance. For example, an excipient can provide form or
consistency, or act as a diluent. Suitable excipients include but
are not limited to stabilizing agents, wetting and emulsifying
agents, salts for varying osmolarity, encapsulating agents,
buffers, and skin penetration enhancers. Excipients as well as
formulations for parenteral and nonparenteral drug delivery
including nasal formulations are set forth in Remington's
Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).
[0217] Generally, these compositions are formulated for
administration by inhalation or nasal application or by injection,
e.g., intraperitoneally, intravenously, subcutaneously,
intramuscularly, etc. Accordingly, these compositions are
preferably combined with pharmaceutically acceptable vehicles such
as saline, Ringer's solution, dextrose solution, and the like. The
particular dosage regimen, i.e., dose, timing and repetition, will
depend on the particular individual and that individual's medical
history.
[0218] In some embodiments, more than one antigen(s) may be present
in a composition. Compositions or formulations of the invention may
be liquid dispersions where the carrier liquid is water and groups
of particles provide the dispersion. A plurality of groups can
provide at least two peptide species, at least 3, at least 4, at
least 5, at least 10, at least 20, and usually not more than 50,
not more than 40, not more than 30 peptide species. Such
"cocktails", as they are often denoted in the art, may be
particularly useful in immunizing against pathogens present in
different variants, e.g. HIV, rotavirus, influenza, etc.
Alternatively, such formulations can provide information on
developing a vaccine.
[0219] Generally, the efficacy of administering any of these
compositions is adjusted by measuring any change in the immune
response as described herein, or other clinical parameters.
[0220] In some embodiments, the invention provides compositions
comprising polypeptides as described herein and an adjuvant whereby
the polypeptide(s)/adjuvant are co-administered. The immunogenic
composition may contain an amount of an adjuvant sufficient to
potentiate the immune response to the immunogen. Adjuvants are
known in the art and include, but are not limited to, oil-in-water
emulsions, water-in oil emulsions, alum (aluminum salts), liposomes
and microparticles including but not limited to, polystyrene,
starch, polyphosphazene and polylactide/polyglycosides.
[0221] Other suitable adjuvants also include, but are not limited
to, MF59, DETOX.TM. (Ribi), squalene mixtures (SAF-1), muramyl
peptide, saponin derivatives, mycobacterium cell wall preparations,
monophosphoryl lipid A, mycolic acid derivatives, nonionic block
copolymer surfactants, Quil A, cholera toxin B subunit,
polyphosphazene and derivatives, and immunostimulating complexes
(ISCOMs) such as those described by Takahashi et al. (1990) Nature
344:873-875, as well as, lipid-based adjuvants and others described
herein. For veterinary use and for production of antibodies in
animals, mitogenic components of Freund's adjuvant (both complete
and incomplete) can be used.
[0222] In some embodiments, the plurality of groups of microspheres
or particles of defined size comprising distinct antigen species
described herein can be administered in conjunction with one or
more immunomodulatory facilitators. Thus, the invention provides
compositions comprising plurality of microspheres of defined size
comprising distinct antigen species and an immunomodulatory
facilitator. As used herein, the term "immunomodulatory
facilitator" refers to molecules which support and/or enhance
immunomodulatory activity. Immunomodulatory facilitators include,
but are not limited to, co-stimulatory molecules (such as
cytokines, chemokines, targeting protein ligand, trans-activating
factors, peptides, and peptides comprising a modified amino acid)
and adjuvants (such as alum, lipid emulsions, and
polylactide/polyglycolide microparticles).
[0223] The following excipients are commonly present in
compositions to generate an immune response such as vaccine
preparations. Aluminum salts or gels are added as adjuvants.
Adjuvants are added to promote an earlier, more potent response,
and more persistent immune response to the vaccine; they allow for
a lower vaccine dosage. Antibiotics are added to some vaccines to
prevent the growth of bacteria during production and storage of the
vaccine. Egg protein is present in influenza and yellow fever
vaccines as they are prepared using chicken eggs. Other proteins
may be present. Formaldehyde is used to inactivate bacterial
products for toxoid vaccines. Formaldehyde is also used to kill
unwanted viruses and bacteria that might contaminate the vaccine
during production. Monosodium glutamate (MSG) and 2-phenoxyethanol
are used as stabilizers in a few vaccines to help the vaccine
remain unchanged when the vaccine is exposed to heat, light,
acidity, or humidity. Thimerosal is a mercury-containing
preservative that is added to vials of vaccine that contain more
than one dose to prevent contamination and growth of potentially
harmful bacteria.
[0224] Many vaccines need preservatives to prevent serious adverse
effects. Several preservatives are available, including thiomersal,
phenoxyethanol, and formaldehyde. Thiomersal is more effective
against bacteria, has better shelf life, and improves vaccine
stability, potency, and safety, but in the U.S., the European
Union, and a few other affluent countries, it is no longer used as
a preservative in childhood vaccines, as a precautionary measure
due to its mercury content. Although controversial claims have been
made that thiomersal contributes to autism, no convincing
scientific evidence supports these claims.
Administration and Assessment of the Immune Response
[0225] The plurality of groups of microspheres or particles of the
invention of defined size, where each group of particles defines a
distinct antigen species composition can be administered in
combination with other pharmaceutical and/or immunogenic and/or
immunostimulatory agents and can be combined with a physiologically
acceptable carrier thereof.
[0226] As with all immunogenic compositions, the immunologically
effective amounts and method of administration of the particular
formulation can vary based on the individual, what condition is to
be treated and other factors evident to one skilled in the art.
Factors to be considered include the antigenicity, route of
administration and the number of immunizing doses to be
administered. Such factors are known in the art and it is well
within the skill of immunologists to make such determinations
without undue experimentation. A suitable dosage range is one that
provides the desired modulation of immune response to the antigen.
Generally, a dosage range may be, for example, from about any of
the following, referencing the amount of peptide in a dose
exclusive of carrier: 0.01 to 100 .mu.g, 0.01 to 50 .mu.g, 0.01 to
25 .mu.g, 0.01 to 10 .mu.g, 1 to 500 .mu.g, 100 to 400 .mu.g, 200
to 300 .mu.g, 1 to 100 .mu.g, 100 to 200 .mu.g, 300 to 400 .mu.g,
400 to 500 .mu.g. Alternatively, the doses can be about any of the
following: 0.1 .mu.g, 0.25 .mu.g, 0.5 .mu.g, 1.0 .mu.g, 2.0 .mu.g,
5.0 .mu.g, 10 .mu.g, 25 .mu.g, 50 .mu.g, 75 .mu.g, 100 .mu.g.
Accordingly, dose ranges can be those with a lower limit about any
of the following: 0.1 .mu.g, 0.25 .mu.g, 0.5 .mu.g and 1.0 .mu.g;
and with an upper limit of about any of the following: 250 .mu.g,
500 .mu.g and 1000 .mu.g. The absolute amount given to each patient
depends on pharmacological properties such as bioavailability,
clearance rate and route of administration.
[0227] The effective amount and method of administration of the
particular formulation can vary based on the individual patient and
other factors evident to one skilled in the art. Routes of
administration include but are not limited to all types of
injection including IV and IM, topical, dermal, transdermal,
transmucosal, nasal, oral, epidermal, parenteral, gastrointestinal,
and naso-pharyngeal and pulmonary, including transbronchial and
transalveolar. The absolute amount given to each patient depends on
pharmacological properties such as bioavailability, clearance rate
and route of administration.
[0228] Parenteral routes of administration include but are not
limited to electrical (iontophoresis) or direct injection such as
direct injection into a central venous line, intravenous,
intramuscular, intraperitoneal, intradermal, or subcutaneous
injection. Compositions suitable for parenteral administration
include, but are not limited, to pharmaceutically acceptable
sterile isotonic solutions. Such solutions include, but are not
limited to, saline and phosphate buffered saline for injection of
the compositions.
[0229] Naso-pharyngeal and pulmonary routes of administration
include, but are not limited to, inhalation, transbronchial and
transalveolar routes. The invention includes compositions suitable
for administration by inhalation including, but not limited to,
various types of aerosols for inhalation, as well as powder forms
for delivery systems. Devices suitable for administration by
inhalation of include, but are not limited to, atomizers and
vaporizers. Atomizers and vaporizers filled with the powders are
among a variety of devices suitable for use in inhalation delivery
of powders.
[0230] The methods of producing suitable devices for injection,
topical application, atomizers and vaporizers are known in the art
and will not be described in detail.
[0231] The above-mentioned compositions and methods of
administration are meant to describe but not limit the methods of
administering the compositions of the invention. The methods of
producing the various compositions and devices are within the
ability of one skilled in the art and are not described in detail
here.
[0232] Analysis (both qualitative and quantitative) of the immune
response to the subject compositions can be by any method known in
the art, including, but not limited to, measuring antigen-specific
antibody production (including measuring specific antibody
subclasses), activation of specific populations of lymphocytes such
as CD4+ T cells or NK cells, production of cytokines such as
IFN.gamma., IL-2, IL-4, IL-5, IL-10 or IL-12 and/or release of
histamine. Methods for measuring specific antibody responses
include enzyme-linked immunosorbent assay (ELISA) and are well
known in the art. Measurement of numbers of specific types of
lymphocytes such as CD4+ T cells can be achieved, for example, with
fluorescence-activated cell sorting (FACS). Serum concentrations of
cytokines can be measured, for example, by ELISA. These and other
assays to evaluate the immune response to an immunogen are well
known in the art. See, for example, Selected Methods in Cellular
Immunology (1980) Mishell and Shiigi, eds., W.H. Freeman and
Co.
[0233] In some instances, a Th1 or Th2-type response is stimulated,
i.e., elicited and/or enhanced. With reference to the invention,
stimulating a Th1 or Th2-type immune response can be determined in
vitro or ex vivo by measuring cytokine production from cells
treated with a composition of the invention as compared to those
treated with a conventional vaccine. Methods to determine the
cytokine production of cells include those methods described herein
and any known in the art. The type of cytokines produced in
response to treatment indicates a Th1-type or a Th2-type biased
immune response by the cells. As used herein, the term "Th1-type
biased" cytokine production refers to the measurable increased
production of cytokines associated with a Th1-type immune response
in the presence of a stimulator as compared to production of such
cytokines in the absence of stimulation. Examples of such Th1-type
biased cytokines include, but are not limited to, IL-2, IL-12, and
IFN-.gamma.. In contrast, "Th2-type biased cytokines" refers to
those associated with a Th2-type immune response, and include, but
are not limited to, IL-4, IL-5, and IL-13. Cells useful for the
determination of activity include cells of the immune system,
primary cells isolated from a host and/or cell lines, usually APCs
and lymphocytes.
Delivery Systems
[0234] Tattoo systems can be used to deliver vaccine compositions
to a patient. Tattoo systems are generally hand held devices that
comprise one or more tattoo needles operably linked to a tattoo ink
delivery system and one or more reservoirs of tattoo ink. Tattoo
systems generally function by moving an actuator in a reciprocating
motion that drives a tattoo needle into the skin of the patient,
pushing the tattoo ink into the skin and depositing it there.
Tattoo systems generally allow the user to control, e.g., the depth
of penetration of the tattoo needle into the skin, the speed of the
needle, and the force of application of the needle against the
skin. Many tattoo systems are known in the art, and include, for
example, tattoo systems used to create decorative tattoos on the
skin of a subject, and medical tattoo systems, typically used in
the field of radiation therapy to identify landmarks on a patient's
skin so that subsequent doses of radiation treatment can be
administered to the same location on a patient's body. Medical
tattoo systems also include systems for drug delivery, e.g., for
delivery of medicines to the skin of a patient. Exemplary tattoo
systems include those disclosed in, e.g., U.S. Pat. Nos. 4,159,659,
6,626,927 and 6,685,719. Exemplary tattoo systems include rotary
tattoo machines, coil tattoo machines, liner tattoo machines,
shader tattoo machines, and color/saturation tattoo machines, any
of which may be adapted for use with the compositions and methods
of the present disclosure.
Cosmetic Tattoo Systems
[0235] An exemplary tattoo system may comprise a frame assembly, an
electromagnet assembly, an armature assembly, an interrupter switch
assembly, a needle holder assembly, a needle guide or handpiece
assembly, and a needle assembly, of which there may be several
different types from which the operator selects one for performing
particular tattooing functions. The needle assembly may comprise an
elongated linear needle shaft and one or more small needles
soldered to the end of the needle shaft opposite the end which is
fitted in the needle holder block. Different tattooing needle
assemblies may be used interchangeably, depending upon the nature
of the work which is to be performed. Two commonly used tattooing
needle assemblies are the outliner needle assembly and the shader
needle assembly. In the outliner needle assembly, five needles are
soldered together in parallel in a perfect circle. In the shader
assembly, six needles are aligned side-by-side in line abreast and
soldered together with adjacent needles contacting each other.
[0236] In use, an operator holds the tattooing device in one hand
by a hand piece or handle. In some embodiments, the needles, or
other selected needle group, are dipped in tattoo ink to prepare
for application of the tattoo ink to the patient's skin. In some
embodiments, tattoo ink is fed to the tattoo needle from a tattoo
ink reservoir or cartridge in a continuous or semi-continuous
manner. In some embodiments, the apparatus is energized by closing
a foot switch that is provided in order to leave the operator's
hands free for manipulation of the tattoo device. Once electric
power is applied to the device, the electromagnets are energized
and attract the armature bar. The attraction of the armature bar to
the electromagnets separates the contacts of the interrupter
switch, thereby de-energizing the electromagnets. The armature bar
is then returned to the normal position by the armature support
spring and the interrupter switch is again closed, restoring the
continuity of the circuit to the electromagnets. The electromagnets
thereby are alternately energized and de-energized repeatedly as
long as the foot switch is closed. The armature bar oscillates and
its oscillation causes the needle assembly to reciprocate in the
needle guide assembly. The needle assembly, including the needle
shaft and needles, is preferably made of stainless steel or other
suitable stainless metal which may be sterilized.
Surgical Tattoo Systems
[0237] In addition to the embodiments described above, the present
disclosure generally provides surgical tattooing systems and
methods that can be used to administer a vaccine composition to a
patient. The following discussion includes a general description of
the instrumentation utilized in applying a surgical tattoo followed
by a description of a method or protocol utilizing the
instrumentation.
[0238] A surgical tattoo ink dispensing instrument includes
essentially three elements, namely, an ink cartridge, a leading
cap, and a trailing cap. The ink cartridge defines an internal
cavity that accommodates the sterile ink. The ink cartridge may
have a generally frustoconical outer shape or configuration with an
enlarged trailing end that gradually tapers to a smaller leading
end. The leading end defines a narrowed distribution port that has
an internal lumen communicating with the internal cavity of the ink
cartridge.
[0239] The trailing end of the ink cartridge includes a
circumferential collar to which a trailing cap is mounted. In one
embodiment, the trailing cap includes a transverse outer wall which
is joined to a circumferential collar of the ink cartridge by
conventional means, including, for example adhesives, cements,
and/or snap fit threaded means. In one embodiment, the trailing cap
is joined to the ink cartridge through an ultrasonic welding
technique. The trailing cap may define an internal circumferential
stop that is received within the trailing end of the ink cartridge
in sealing engagement therewith.
[0240] The trailing end cap defines an elongated length that
facilitates functioning of the trailing end cap as a handle. In one
embodiment, in use the operator holds the end cap in a manner
similar to that of a writing implement, thus providing ease of
control and a degree of familiarity to the operator.
[0241] The leading cap is placed on the ink cartridge prior to
loading of the ink and preferably includes an internal stop that is
received within the lumen of the port to fluidly seal the port at
this end. The leading cap further defines an internal
circumferential wall that is mounted about the port and preferably
dimensioned so as to frictionally engage the port to thereby retain
the end cap on the ink cartridge during assembly and shipping.
[0242] In some embodiments, the tattoo ink filled within the ink
cartridge comprises a vaccine to be delivered to a patient. In some
embodiments, the tattoo ink filled within the ink cartridge
comprises a suitable tattoo ink, e.g., a selectively activatable
tattoo ink, for use in tracking patients who have been vaccinated
with a compound of the present disclosure. Any of a variety of
suitable inks and/or vaccine compositions may be used in
conjunction with the tattooing systems and methods discussed
herein.
[0243] The components of the tattooing instrument may be
fabricated, e.g., from a suitable polymeric material by known
injection molding techniques. Ink cartridges may have some rigidity
to minimize deformation thereof whereby the tattoo ink is gravity
fed through a distribution port. Alternatively, an ink cartridge
may be flexible to be squeezed in a manner to force the tattoo ink
through the distribution port. In some embodiments, the trailing
end cap is rigid.
[0244] Exemplary dimensions of the instrument may be as follows:
length of trailing end cap: 2-3 inches, preferably about 2.5
inches; length of ink cartridge: 1.5-2.1 inches, preferably about
1.8 inches; diameter of trailing end cap: 0.250-0.450 inches,
preferably about 0.350 inches. Other dimensions may be desirable
based on the contemplated use of the device.
[0245] In one method of manufacture, the leading cap is mounted to
the ink cartridge and the ink cartridge is filled with sterile
tattoo ink. Thereafter, the trailing cap is then mounted to the ink
cartridge and sealed through the ultrasonic weld joint. Thus the
entire device is hermetically sealed, sterile and filled with the
appropriate tattoo ink. This device can be prepackaged and shipped
and delivered to the end user.
[0246] The ink dispensing instruments described above are intended
for use in conjunction with a suitable tattoo needle, which may be
secured to the device through any number of known conventional
means. For example, the needle can be mounted about the distal port
and connected therewith with threaded means on the needle as
internal threads that threadably engage corresponding threads of a
cartridge, a bayonet coupling, a frictional engagement, etc.
Alternatively, the needle is not required and the ink from the ink
cartridge can be dispensed onto a needle point which is
subsequently applied to the skin of the patient for tattooing.
[0247] In some embodiments, the trailing cap is eliminated. The ink
cartridge may have an elongated portion or handle adjacent its
trailing end and integrally attached thereto. The handle may
include recessed grooves that accommodate the fingers of the user.
The elongated portion of the handle enables the operator to hold
the instrument in a manner similar to using a pencil or pen. This
provides the user with a familiar feel that enhances control when
applying the tattoo. In some embodiments, this instrument defines a
length ranging from 4.00-4.50 inches, preferably about 4.25
inches.
[0248] In some embodiments, the device includes an ink cartridge
defining a generally bulbous shape which is rounded at its proximal
end. The distal end of the ink cartridge includes a distal port and
has an end cap that is mounted to the distal port in the manner
discussed above. The ink cartridge may comprise two components,
namely, the ink base cartridge and a nozzle that defines the distal
port and is ultrasonically welded to the base to connect the two
components. The bulbous ink cartridge facilitates holding of the
instrument between the index finger, middle finger, and the thumb,
and provides a greater area to facilitate squeezing or deformation
of the ink cartridge to expel the sterile tattoo ink.
Surgical Tattooing Protocol
[0249] A surgical tattooing protocol for use with the
instrumentation described above will now be discussed. First, a
prepackaged ink dispensing instrument is provided and opened by
tattooing personnel. A sterilized tattooing needle is mounted to
the ink cartridge as discussed hereinabove. The needle is then
applied to the skin of the patient. Next, the ink is dispensed or
squeezed (or if gravity fed no squeezing is required) from the
cartridge to deliver the ink through the distal port into the
needle and then directed into the skin of the patient. After use,
the needle and ink cartridge are discarded. In an alternate
arrangement, a tattooing needle is not mounted to the ink
cartridge, but, instead, is introduced to the skin. Thereafter, the
tattoo ink is applied to the outer surface of the needle to travel
along the needle and into the tissue.
[0250] It is contemplated that, in some embodiments, the dispensing
instrument of the present disclosure will be individually wrapped
in a sterile packet and boxed. The packets may be attached to each
other, and, in such a case, an operator could pull off one packet
at a time as needed. See, e.g., the dispensing system disclosed in
U.S. Pat. Nos. 5,358,140 and 5,282,349, the contents of each being
incorporated herein by reference. The instruments may be used for,
e.g., safe and sterile application of tattoo ink to the skin of a
patient for purposes of, e.g., administering a vaccine to a patient
and/or marking a patient with an identifying marking. Other uses
are contemplated as well.
[0251] In some embodiments, a tattoo system may be preloaded with
permanent or semi-permanent tattoo ink. The preloaded instrument
could be used to tattoo the skin of a patient and thereafter
activated to deliver the tattoo ink to appropriately tattoo the
patient for purposes of, e.g., tracking and/or identifying the
patient following administration of a vaccine composition. After
use, the instrument could be disposed of, although it is envisioned
that in some embodiments the device could be sterilized for
subsequent use(s).
[0252] The present disclosure thus contemplates the surgical
tattooing methods and systems discussed above. The system assures
that sterile tattoo ink is used for each individual patient.
[0253] There are several new delivery systems in development to
make vaccine delivery more efficient. Methods include liposomes and
ISCOM (immune stimulating complex). Other vaccine delivery
technologies have resulted in oral vaccines. A polio vaccine was
developed and tested by volunteer vaccinations with no formal
training; the results were positive in that the ease of the
vaccines increased dramatically. With an oral vaccine, there is no
risk of blood contamination. Oral vaccines are likely to be solid
which have proven to be more stable and less likely to freeze; this
stability reduces the need for a "cold chain": the resources
required to keep vaccines within a restricted temperature range
from the manufacturing stage to the point of administration, which,
in turn, will decrease costs of vaccines.
[0254] A microneedle approach may be used, where the microneedle,
which is "pointed projections fabricated into arrays that can
create vaccine delivery pathways through the skin". Microneedles
(MN), as used herein, refers to an array comprising a plurality of
micro-projections, generally ranging from about 25 to about 2000
.mu.m in length, which are attached to a base support. An array may
comprise 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5 or more
microneedles, and may range in area from about 0.1 cm.sup.2 to
about 100 cm.sup.2. Application of MN arrays to biological
membranes creates transport pathways of micron dimensions, which
readily permit transport of macromolecules such as large
polypeptides. The microneedle array may be formulated as a
transdermal drug delivery patch. MN arrays can alternatively be
integrated within an applicator device which, upon activation, can
deliver the MN array into the skin surface, or the MN arrays can be
applied to the skin and the device then activated to push the MN
through the skin.
METHODS OF THE INVENTION
[0255] The invention also includes methods of modulating an immune
response comprising administering an immunogenic formulation as
described herein to an individual in an amount sufficient to
modulate the immune response. Generally, the individual is in need
of, or will be in need of, such modulation, due, for example, for a
disease condition or being at risk of developing a disease
condition. Examples of disease conditions include, but are not
limited to, allergy, cancer, infectious diseases (such as viral or
bacterial infection).
[0256] Methods of the invention include manufacturing particles of
a biocompatible polymer having individual chemical species within
the particle and then manufacturing groups of particles to create
formulations and using those formulations to modulate an immune
response within a subject, or, alternatively, using those
formulations to study the immune system of a subject to obtain
information in the development of a formulation to modulate an
immune response in a subject.
[0257] A method of the invention includes administering a
formulation of the invention to a patient such as a human patient
and allowing the patient to generate an immune response to the
formulation. The method may be carried out wherein a single
formulation is administered to a patient wherein the single
formulation is comprised of a plurality of groups of particles
which are comprised of a biocompatible polymer and a chemical
species such as an amino acid sequence which is an antigen specific
to a pathogen. The particles within a given group may be identical
and the particles within different groups are different with
respect to the chemical species. The particles within all of the
groups are within a size range in accordance with the invention
such as a size range of 10 microns.+-.20% to 25 microns.+-.20%, or
13 microns to 17 microns in diameter.+-.20%.
[0258] The methodology is intended to be carried out wherein the
patient such as a human patient has not been previously exposed to
any of the chemical species within the formulation administered to
the patient. Thus, the method of the invention is generally not
intended as a "booster" to supplement the immune response generated
by a prior exposure of one or more of the antigens within the
formulation. An aspect of the invention includes administering a
formulation of the invention to a patient such as a human patient
by a means such as injection, nasal spray or nasal drops and
allowing the formulation to generate an immune response. The
patient is generally a human patient believed to be at risk for an
infection such as a viral or bacterial infection. Preferably, the
patient has not been previously exposed artificially or naturally
to pathogens (naturally) or antigens (artificially) which
correspond to the antigens of the formulation of the invention. The
reasoning as to why the method should be carried out as an initial
and only exposure is that the concept of the invention relates at
least in part to modulating or eliminating problems associated with
immunodominance.
System of Tattoo Marking
[0259] The present disclosure generally provides methods of
vaccinating a subject and/or identifying the vaccinated subject
through the use of one of more identifying markings that are
applied to the subject's skin.
[0260] For example, in some embodiments a vaccine composition is
formulated for delivery to a patient through the use of a tattoo
system. First, the vaccine composition is administered to the
patient by tattooing the patient with the vaccine composition,
thereby administering the vaccine composition to the patient's
skin. In some embodiments, the patient is subsequently tattooed
using a tattoo ink that contains a visible or selectively
activatable tattoo ink in order to create an identifying marking on
the patient that can be used for identification purposes. For
example, the patient may be tattooed with an identifying letter,
number, symbol, or other device that identifies the patient based
on the particular vaccine composition that was administered to the
patient. Application of the identifying tattoo to the patient's
skin allows clinical personnel to identify the particular vaccine
composition that was administered to the patient. Following
administration of the vaccine and application of the tattoo, the
patient can be identified, and the patient's response to the
vaccine can be tracked for clinical purposes.
Administration of a Vaccine Composition and Application of an
Identifying Tattoo at the Same Time
[0261] In some embodiments, a vaccine composition may be formulated
to contain a tattoo ink that is visible or selectively activatable,
and can thus be used to create an identifying marking on the
patient at the same time that the vaccine composition is
administered to the patient.
[0262] For example, in some embodiments, a vaccine composition is
formulated to contain a visible or selectively activatable tattoo
ink. The vaccine composition is loaded into a suitable tattoo
device and the device is used to tattoo an identifying marking on
the patient. Application of the tattoo ink administers the vaccine
to the patient, while at the same time creating an identifying
marking that can be used by clinical personnel to identify the
particular vaccine composition that was administered to the
patient. Following application of the tattoo, the patient can be
identified, and the patient's response to the vaccine can be
tracked for clinical purposes.
Class I and Class II Epitopes
[0263] In one embodiment of the invention, only a single antigen
species is present in a single particle or sphere. However, it is
possible to include more antigen species in certain situations. As
explained below different antigen species can be present in one
particle when each species provides epitopes that are presented by
different classes of MHC protein.
[0264] First it should be understood that an epitope, also known as
antigenic determinant, is the part of an antigen that is recognized
by an antigen receptor of the immune system, specifically by
soluble antibodies, antibodies present on the surface of B cells,
or T cells. Although epitopes are usually thought to be derived
from non-self proteins, sequences derived from the host that can be
recognized are also classified as epitopes, as well as
non-proteinaceous antigens. The present invention is generally
concerned with epitopes antigenic molecules from pathogens and more
specifically pathogens such as bacteria and viruses that infect
humans, and still more specifically those having an amino acid
sequence limited to 8 to 12 amino acids
[0265] In humans the Major Histocompatibility Complex (MHC) is a
set of molecules displayed on cell surfaces, which are integral to
the antigen presentation machinery responsible for lymphocyte
recognition and "antigen presentation". The interaction between MHC
molecules, antigen receptors and co-receptors controls mammalian
immune responses, and provides a mechanism for distinguishing
"self" and "non-self". Both Class I and Class II MHC molecules
belong to a group of molecules known as the Immunoglobulin
Supergene Family, which includes immunoglobulin, T-cell receptors,
CD4, CD8, and others.
[0266] Class I polypeptide molecules are composed of two
polypeptide chains; one encoded by the MHC region and another
(.beta.2-microglobulin) that is encoded elsewhere. The MHC-encoded
polypeptide is about 350 amino acids long and glycosylated, giving
a total molecular weight of about 45 kDa. This polypeptide folds
into three separate domains called alpha-1, alpha-2 and alpha-3.
.beta.2-microglobulin is a 12 kDa polypeptide that is
non-covalently associated with the alpha-3 domain. Between the
alpha-1 and alpha-2 domains lies a region bounded by a beta-pleated
sheet on the bottom and two alpha helices on the sides. This region
is capable of binding (via non-covalent interactions) a small
peptide or epitope of about 8 to 12 amino acids. Included in the
human class I proteins, also referred to as HLA, are HLA-A, HLA-B,
HLA-C, and (.beta..sub.2-microglobulin.
[0267] Large protein antigens are cleaved within antigen presenting
cells into small peptides, which may be from 8-12 amino acids in
length. The small peptides that have an appropriate affinity for
the MHC Class I protein (see, for example, Liao et al, (2011) PLoS
One. 6(9):e25055, "Predicting peptide binding affinities to MHC
molecules using a modified semi-empirical scoring function.") and
are complexed with the MHC protein, and are then transported to the
cell surface, where they combine with protein components, i.e.
coreceptors in the cellular membrane to form a complex that the
T-cell recognizes. A CD8+ cytotoxic T-cell, for example, can be
activated when the T cell antigen receptor on its surface binds to
cognate epitopes complexed with class I mHC proteins on the surface
of a cell, which can include without limitation antigen presenting
cells.
[0268] In accordance with the invention, a single particle will
generally only include one peptide or epitope that is presented by
class I molecules present on the cell of interest. Such a peptide
or epitope is referred to here as a class I peptide or a class I
epitope.
[0269] Class II molecules are composed of two polypeptide chains.
These polypeptides (alpha and beta) are about 230 and 240 amino
acids long, respectively, and are glycosylated, giving molecular
weights of about 33 kDa and 28 kDa. These polypeptides fold into
two separate domains; alpha-1 and alpha-2 for the alpha
polypeptide, and beta-1 and beta-2 for the beta polypeptide.
Between the alpha-1 and beta-1 domains lies a region very similar
to that seen on the class I molecule. This region, bounded by a
beta-pleated sheet on the bottom and two alpha helices on the
sides, is capable of binding (via non-covalent interactions) a
small epitopes or peptide of about 8 to 12 amino acids, although a
longer peptide can extend outside of the binding pocket (see, for
example Fleckenstein et al. (1999) Semin Immunol. 11(6):405-16,
"Quantitative analysis of peptide-MHC class II interaction"). These
small peptides can be moved to the surface of the cell to form a
complex recognizable by T-cells in a similar fashion to that
described for Class I molecules as described above. Included in the
human class II proteins are HLA-DP.alpha., HLA-DP.beta.,
HLA-DQ.alpha., HLA-DQ.beta., HLA-DR.alpha. and HLA-DR.beta.. These
small peptides or epitopes are referred to here as class II
peptides or class II epitopes.
[0270] While class I and class II molecules appear somewhat
structurally similar and both present antigen to T-cells, their
functions are distinct. Class I proteins are present on virtually
all nucleated cells in the body, while class II protein expression
is generally restricted to antigen presenting cells, e.g. dendritic
cells, macrophages and B cells. Class I polypeptide molecules
present antigen to cytotoxic T-cells (CTLs) in the context of CD8
co-receptor proteins, while class II polypeptide molecules present
antigen to helper T-cells (TH-cells) in the context of CD4
co-receptor proteins.
[0271] Class I polypeptide molecules, which are present on the
surface of virtually all nucleated mammalian cells, typically
present "endogenous" antigens, which are fragments of proteins
present within the cell. Examples include viral proteins or tumor
proteins that are being actively expressed by the cell.
Presentation of such antigens can indicate internal cellular
alterations that if not contained could spread throughout the body.
Hence, destruction of these cells by CTLs is advantageous to the
body as a whole.
[0272] Class II polypeptide molecules present on APCs usually
complex with "exogenous" antigens, which are not necessarily being
expressed by the APC. Exogenous antigens are typically present in
the extracellular space and are presented after being engulfed and
processed by the APC. It may be noted that a viral protein could be
endogenous to a cell infected by the virus, or exogenous to a cell
that is exposed to a viral particle. The presentation of an antigen
(class II peptide) by a class II polypeptide molecule usually
stimulates T helper cells, which can, in turn, activate effector
cells such as B cells (to produce antibodies), or CTLs.
[0273] For the case of the present invention, an immune response
with memory is desired after a vaccination event, with or without
the administration of a booster dose. Although the class-I epitopes
administered in the micro particles provide sufficient information
for activation of relevant T-cells to fight the infection of
interest, an immune response from class-II epitopes may also be
advantageous. In particular, a CD4 T-cell response to a "generic"
epitope not specific for a CTL epitope may facilitate producing an
anamnestic immune response to the CTL epitope. Formulations of the
invention may include particles with multiple copies of one class I
epitope and multiple copies of one class II epitope. In general, a
single particle of the invention does not include multiple
different class I epitopes.
[0274] An aspect of the invention is a particle comprised of an
inert biocompatible polymer and an active ingredient which consists
only of (a) multiple copies of a class I epitope and (b) multiple
copies of a class II epitope. The particle has a size of about 12
to 18 microns or 15 microns.+-.20%. The class I epitope and the
class II epitope consists of 8 to 20 amino acids, or 8 to 12 amino
acids or about 15 amino acids.
EXAMPLES
[0275] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
[0276] In Example 1 10 micron diameter spherical particles 3, 3',
3'', etc. comprised of poly(lactic-co-glycolic acid) (PLGA) are
used. The particles had substantially the same diameter.+-.10% or
less. The particles were placed in a solution containing human
dendrocytes 1 and 2. Photos were taken of the cells prior to (FIG.
1A), during (FIG. 1B) and (FIG. 1C) and after (FIG. 1D) the cells 1
and 2 consumed the particles. The particles 3, 3', 3'', etc. were
produced using a process as described within U.S. Pat. No.
6,116,516.
Example 2
[0277] In Example 1 15 micron diameter spherical particles 3, 3',
3'', etc. comprised of poly(lactic-co-glycolic acid) (PLGA) are
used. The particles had substantially the same diameter.+-.10% or
less. The particles were placed in a solution containing human
dendrocytes 1 and 2. Photos were taken of the cells prior to (FIG.
2A), during (FIG. 2B) and (FIG. 2C) and after (FIG. 2D) the cells 1
and 2 consumed the particles. The particles 3, 3', 3'', etc. were
produced using a process as described within U.S. Pat. No.
6,116,516.
[0278] Examples 1 and 2 show how groups of particles can be
administered (placed in contact with dendrocytes) and used to
determine the size of particles which the dendrocytes of the immune
system readily consume. The results of Examples 1 and 2 indicate
that for these dendrocytes, particles which are 10 microns in
diameter are sufficiently small that multiple particles can be
consumed by a single dendrocyte. The 15 micron particles of Example
2 indicate that, for these dendrocytes, only a single particle of
15 microns in diameter can be consumed. Thus, these two Examples
indicate, for these dendrocytes, that the particles with a diameter
of 15 microns can be used to present a single chemical species, and
thereby reduce or eliminate immunodominance issues which might be
created when multiple species are simultaneously presented by
dendrocytes.
[0279] The formula for the volume of a sphere is
(4/3).times.pi.times.(radius).sup.3. The radius of the 10 micron
sphere is 5 and 5.sup.3 is 125. The radius of the 15 microspheres
is 7.5 and 7.5.sup.3 is 421.815. Thus, a relatively small increase
in the diameter of a sphere makes a large increase in the volume of
the sphere. The sphere volume is important in terms of the size of
the particle that can undergo dendrocyte phagocytosis.
Example 3
[0280] Synthesis of antigen containing microspheres. Microspheres
of defined size, and containing a single peptide species were
synthesized as described in Table 2 below.
TABLE-US-00002 TABLE 2 Reagent Name Supplier Cat. No. Purity
Resomer 502H Boehringer Mannheim 502H 99% D-(+)-Mannose Sigma M6020
98% CMV pp65 peptide* American Peptide 305264 95%
Phosphate-buffered Sigma D8537 100% saline (PBS) Acetone Sigma
270725 .gtoreq.99.9% .sup. *Note: Any peptide may be used in the
synthesis.
[0281] CMV pp65 peptide was solubilized in PBS at 25 mg/ml
(hereafter Reagent A; stored at 4 C). Mannose was solubilized in
PBS at 200 mgs+400 uL PBS (hereafter Reagent B; stored at room
temperature).
[0282] For 5 mls of formulation: a) Place 200 mgs of Resomer 502H
in glass vial; b) Add 5.0 mL acetone and mix by rocking until
Resomer is completely solubilized; c) Place vial in sonicator; d)
During sonication, add 80 uL of Reagent A slowly, drop-wise; e)
During sonication, add 20 uL of Reagent B slowly, drop-wise; f) Cap
vial tightly and continue to sonicate.
[0283] The microspheres were formulated for use in cell cultures by
suspension in appropriate culture medium. For use in pharmaceutical
formulations the culture medium may be substituted with an
appropriate excipient, e.g. normal saline, PBS, and the like.
[0284] In such formulations, 1 mg of the antigen containing
microspheres is resuspended at a concentration (w/v) equal to 1
milligram/100 microliters in sterile tissue culture medium, and
mixed well before use by flicking or sonication. The microspheres
will settle within minutes so they must be re-mixed directly before
using.
[0285] For phagocytosis experiments, 10 .mu.l of resuspended
microspheres were added to each well of as 24 well plate. In a 96
well plate, 2 .mu.l t of resuspended microspheres are used.
Example 4
Assessment of Antigen Presentation
[0286] Preparation of Dendritic Cells from peripheral blood
mononuclear cells (PBMC). Frozen PBMC cells were obtained from
Cellular Technology Limited, from volunteer donors known to have
been exposed to CMV, and to be HLA-A02. The volunteer's antigen
presenting cells were known to be capable of presenting HCMV pp65
(NLVPMVATV) CMV epitope to T lymphocytes. Human PBMC (Peripheral
Blood Mononuclear Cells) isolated from leukopacks and frozen in
serum-free CTL-Cryo.TM. medium. These leukopacks were ethically
collected from healthy donors tested for: HBsAg, HBcAb, HCV, HTLV
I/II, and STS by serology; as well as HIV I, HCV, and WNV by
NAT.
[0287] Cells were thawed and plated at a concentration of
1.times.10.sup.5 monocytes/well of a 24-well plate in medium
supplemented with GM-CSF at a final concentration of 50 ng/ml and
IL-4 at a final concentration of 100 ng/ml. Cells were maintained
in culture at 37.degree. C./5% CO.sub.2 for 6 days; and used with 8
days after that point. Optical microscopy confirmed maturation of
monocytes to dendrocytes capable of phagocytosis of 15 micron PLGA
microspheres.
[0288] Presentation of antigen and stimulation of T cells was
assessed by production of human .gamma.-IFN, measured by ELISA.
Mature dendrocytes were mixed with T-Lymphocytes and incubated for
two hours with either free, soluble NLVPMVATV (at 10.sup.-9 M
concentration) or 15 micron PLGA microspheres containing
approximately 2% NLVPMVATV, prepared as described in Example 3.
Production of .gamma.-IFN was measured after 2 hours. The results
are as shown in Table 3, demonstrating that 15 micron microspheres
provide antigen that can be taken up by dendritic cells and
presented to T cells.
TABLE-US-00003 TABLE 3 Material Added To Cell Prep ELISA Optical
Density Unstimulated Cells (no material added) 0.055 Incubated with
free peptide 0.612 incubated with microspheres 0.560
Example 5
[0289] 6-8 week old male C57Bl/6 mice were subjected to
intra-dermal (tail) injection with the H-2Kb-restricted peptides
SIINFEKL (OVA) and RGYVYQGL (VSV). Initial experiments demonstrated
that free epitopes when injected with the adjuvants CpG and
monophosphoryl lipid A (MPL) produced no measureable immune
response when T-Cells harvested 14 days post inoculation were
challenged with the same epitopes in-vitro with an Elispot assay
evaluating an interferon gamma response.
Example 6
[0290] The epitopes as described in Example 5 were encapsulated
into PLGA microspheres as follows:
[0291] PLGA was prepared as a 4% (w/v) solution in acetone
(typically, 200 mg of PLGA would be placed into an ultraclean
borosilicate vial to which 5 mls of acetone would then be added).
The vial was capped securely and placed on a rocker. The PLGA went
completely into solution with gentle, constant agitation over 20
minutes. The peptide of choice was prepared as a 10 mg/ml solution
in water. Mannose was prepared as a 100 mg/ml solution in water.
The CpG ODN was prepared as a 10 mg/mL solution in water.
[0292] The solution of PLGA in acetone was next placed in a
sonicator. Under constant sonication, the peptide was added. The
amount of peptide varied, from 1 microliter (constituting one
percent (1%) of maximal loading in the final microspheres by
weight) to 100 microliters (constituting 100% of maximal loading).
If necessary, an amount of water was then added to bring the total
amount of aqueous solution added to 100 microliters (e.g., if 10
microliters of peptide was added, 90 microliters of water would
then be added). While still under sonication, 50 microliters of
mannose was added, followed by 50 microliters of the CpG ODN. This
complete formulation was then sonicated for an addition five
minutes.
[0293] Complete formulations were used in the Flow Focusing device
as described in U.S. Pat. No. 6,116,516 (incorporated herein by
reference) within one hour of their preparation. After passage
through the Flow Focusing device, the resultant micro-particles
collected were allowed to air dry overnight before harvesting and
any further analysis and/or use in animal experiment
procedures.
[0294] For the animal procedures, approximately 2.5 mgs of
micro-particles were placed in a micro-centrifuge tube. On the day
the animals were to be vaccinated, 112.5 microliters of water+0.1%
Tween 20* was added to each tube, followed by the addition of 12.5
microliters of MPLA in DMSO. The micro-particles were briefly
vortexed to re-suspend them in the liquid, and then 100 microliters
of this suspension was injected into each animal (i.e., the
resultant amount of micro-particles per animal was 2 mgs).
[0295] The Flow Focusing process was used to fabricate
micro-spheres 13 microns in diameter so as to match the diameter of
mouse antigen presenting cells as determined by light microscopy in
preliminary experiments. This was done (a) to maximize the chance
that only one sphere would be phagocytosed by any given antigen
presenting cell and (b) to maximize the payload of epitope
delivered to the antigen presenting cell per phaygocytosis
event.
[0296] Liquid flow rate was set at 8.0 ml/hr. Gas pressure was 70
millibars. Needle ID was 0.006 inch, nozzle ID was 0.008 inch with
0.144 inch length, separation of needle to nozzle entrance was
0.006 inch. The needle is equivalent to the needle 60 of U.S. Pat.
No. 6,116,516 and nozzle internal diameter is equivalent to the
exit opening 68 of U.S. Pat. No. 6,116,516.
[0297] The peptide and other material are dissolved in acetone at a
total concentration of about 5% w/v. The aerosol is generated at
the top of an evaporation column where the wet droplet size is
about 30 micrometers. After passing through an evaporation chamber
(volume 2.7 liters, flow rate 12 l/min) most of the acetone is
removed and the now dry particles pass through a Malvern Spraytech
laser diffraction particle size analyzer, wherein they are
measured.
[0298] After passing through the Spraytech, the particles were
collected in an Anderson Cascade Impactor running at a flow rate of
15 l/min.
[0299] The plot shown in FIG. 6 is based on a 30 minute run shows a
typical data set for this device. Particle size distribution is
stable throughout the run with a mean size of 13 um. The
distribution is further modified upon capture in the cascade
impactor.
Example 7
[0300] 60 mice were intra-dermally injected with PLGA micro-spheres
prepared as described in Example 6 containing escalating
concentrations of OVA or VSV. The immune response was evaluated via
anti-IFN-gamma Elispot assay 14 days after inoculation and the
results as shown in Table 4 below:
TABLE-US-00004 TABLE 4 Sphere Loading As A IF-Gamma response as
Percent of Maximum percent of positive Epitope Possible Loading
Elispot control OVA (SIINFEKL) 1% 16 VSV (RGYVYQGL) 1% 32
Example 8
[0301] Because the 13 micron size of the fabricated micro-spheres
was determined so as to ensure that any given antigen presenting
cell was likely to only phagocytose and process epitopes from one
sphere at a time, it was possible to design an experiment to see if
a second epitope simultaneously loaded into a single microsphere in
excess concentration could inhibit the presentation of the first
loaded and also to examine if such an effect could be mitigated by
only placing only one type of epitope in any given sphere. If two
different sphere populations were given to the same mouse each
loaded with a different epitope at a different concentration by
weight, the large size of the spheres would make it unlikely that
any single antigen presenting cell could phagocytose two spheres at
once, making inhibition of one epitope by another in a single
antigen presenting cell less likely.
[0302] This study was conducted to evaluate whether the addition of
VSV in the same micro-particle with OVA will inhibit presentation
of OVA peptide and thus reduce OVA-specific IFN-gamma and if
placing OVA and VSV in separate micro particles would mitigate this
inhibitory effect. 40 C57Bl/6 mice were inoculated with the
micro-encapsulated epitopes as follows:
[0303] Three groups of 20 mice in each group were vaccinated with 2
mg epitope-loaded PLGA micro-particles by intra-dermal (tail)
injection. 10 Mice in each group were inoculated with OVA and VSV
epitopes in the same micro-sphere and 10 mice in each group were
inoculated with OVA and VSV in separate spheres. An additional two
mice in each 10 mouse test group served as controls receiving
vehicle (PBS/0.1% Tween 20) only.
[0304] Responses to inoculation were determined by Elispot immune
assay performed 14 days after initial injection evaluating
harvested cells for IFN-gamma release on exposure to each of the
two peptides injected and results are shown in Table 5 below:
TABLE-US-00005 TABLE 5 OVA/VSV MICRO- SAME SPHERE OR SPHERE LOADED
DIFFERENT OVA/VSV IF-gamma RATIO SPHERES RESPONSE RATIO 1:1 SAME
SPHERES 77 1:10 SAME SPHERES 49 1:100 SAME SPHERES 26 1:1 DIFFERENT
SPHERES 68 1:10 DIFFERENT SPHERES 52 1:100 DIFFERENT SPHERES 43
[0305] This data was used to calculate the relative improvement in
immune response seen when the epitopes, at the same relative
concentration ratios, are loaded in different spheres compared with
the same spheres as shown in FIGS. 3 and 4 and summarized below in
Table 6:
TABLE-US-00006 TABLE 6 OVA/VSV IMMUNE RESPONSE RATIO: OVA/VSV RATIO
DIFFERENT SPHERES/SAME SPHERES 1:1 .88 1:10 1.06 1:100 1.65
[0306] This data shows that when OVA and VSV epitopes at a ratio of
1:100 are placed in separate micro-spheres, a greater than 50%
reduction in the inhibitory effect seen on OVA presentation by VSV
can be seen when OVA and VSV at the same 1:100 relative amounts are
each placed in different micro-spheres. The results of the groups
of FIG. 3 are compared to the results of the groups of FIG. 4 were
obtained and the ratios of these results are plotted in FIG. 5.
[0307] It is to be understood that this invention is not limited to
the particular methodology, protocols, peptides, animal species or
genera, constructs, and reagents described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
will be limited only by the appended claims.
[0308] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0309] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing, for
example, the reagents, cells, constructs, and methodologies that
are described in the publications, and which might be used in
connection with the presently described invention. The publications
discussed above and throughout the text are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0310] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *